Long acting proteins and peptides and methods of making and using the same

ABSTRACT

Disclosed is a method for refolding a protein or peptide that does not contain essential disulfides and that contains at least one free cysteine residue. Also disclosed are polymer IFN-γ conjugates that have been created by the chemical coupling of polymers such as polyethylene glycol moieties to IFN-γ, particularly via a free cysteine in the protein. Also disclosed are analogs of bioactive peptides that may be used to create longer acting versions of the peptides, including analogs of glucagon, glucagon-like peptide-1 (GLP-1), GLP-2, Gastric inhibitory peptide (GIP), PYY, exendin, ghrelin, gastrin, amylin, and oxyntomodulin.

STATEMENT REGARDING GOVERNMENT INTERESTS

This invention was made in part with government support under Grant Nos.1R43 AI060043, 2R44 AI060043, and 1R43 CA108001 each awarded by theNational Institutes of Health. The government has certain rights in theinvention.

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing submitted as an electronictext file named “4152-18_ST25.txt”, having a size in bytes of 8 kb, andcreated on 14 Dec. 2007. The information contained in this electronicfile is hereby incorporated by reference in its entirety pursuant to 37CFR §1.52(e)(5).

FIELD OF THE INVENTION

This invention generally relates to novel polymer protein or peptideconjugates, and in a preferred embodiment, novel interferon gamma(IFN-γ) conjugates, that have been created by the chemical coupling ofpolymers such as polyethylene glycol moieties to variants of proteinsand peptides, and methods of making and using such conjugates. Theinvention also relates to methods for producing proteins and peptidesthat lack essential disulfides and contain at least one free cysteineresidue, such proteins including, but not limited to, IFN-γ.

BACKGROUND OF THE INVENTION

There is considerable interest on the part of patients and healthcareproviders in the development of low cost, long-acting, “user-friendly”protein therapeutics. Proteins are expensive to manufacture and unlikeconventional small molecule drugs, are not readily absorbed by the body.Therefore, proteins must be administered by injection. Most proteins arecleared rapidly from the body, necessitating frequent, often daily,injections. This is particularly the case for small peptides, whichoften have half-lives on the order of minutes following injections intohumans. Patients dislike injections, which leads to reduced complianceand reduced drug efficacy. The length of time an injected proteinremains in the body is finite and is determined by the protein's sizeand whether or not the protein contains covalent modifications such asglycosylation.

Introduction of proteins into circulation exposes the proteins tonumerous cell types, enzymes and routes of extravasation that contributeto their rapid clearance or catabolism. The protein may be attacked byplasma proteases or bind plasma proteins or cell surface receptors.Either receptor-mediated or less specific binding may result in theuptake of the protein via endocytotic or pinocytotic mechanisms, withthe end result being degradation by lysosomal proteases. Proteins thatavoid capture by these cells may pass out of the circulation via uptakeby the liver, the lymphatic system or renal glomeruli (Sheffield, 2001).

Circulating concentrations of injected proteins change constantly, oftenby several orders of magnitude, over a 24 hour period. Rapidly changingconcentrations of protein agonists can have dramatic downstreamconsequences, at times understimulating and at other timesoverstimulating target cells. Similar problems plague proteinantagonists. These fluctuations can lead to decreased efficacy andincreased frequency of adverse side-effects for protein therapeutics.The rapid clearance of recombinant proteins from the body significantlyincreases the amount of protein required per patient and dramaticallyincrease the cost of treatment. Most protein products currently on themarket require frequent injections, usually multiple times per week.This dosing regimen is painful, inconvenient for the patient, and maynot provide the optimum therapeutic benefit. In the case of a chronicindication such as cancer or an immunodeficiency, treatment could lastfor years. Thus, there is a strong need to develop protein deliverytechnologies that lower the costs of protein therapeutics to patientsand healthcare providers. One solution to this problem is thedevelopment of methods to prolong the circulating half-lives of proteintherapeutics in the body so that the proteins do not have to be injectedfrequently. This solution also satisfies the needs and desires ofpatients for protein therapeutics that are “user-friendly”, i.e.,protein therapeutics that do not require frequent injections.

Many bioactive peptides have been described, including glucagon,glucagon-like peptide-1 (GLP-1), GLP-2, Gastric inhibitory peptide(GIP), PYY, exendin, ghrelin, gastrin, amylin, and oxyntomoldulin. Thesepeptides typically are 10-40 amino acids in length. Methods to developlonger acting forms of these peptides are desired.

In addition, larger proteins, such as interferon-gamma (IFN-γ) are ofhigh interest as therapeutics. IFN-γ was first recognized over 35 yearsago on the basis of its anti-viral activity (Wheelock, 1965). Over theyears, a great deal of information has accumulated that validatesIFN-γ's role in modulating nearly all phases of immune and inflammatoryprocesses. IFN-γ belongs to a family of proteins related bothstructurally and by their ability to protect cells from viral infection.The interferon family has three main members, now designatedinterferon-alpha (IFN-α), interferon-beta (IFN-β), and interferon-gamma(IFN-γ). The latter, which is also known as immune or type IIinterferon, has several properties related to immunoregulation thatmakes it different from the other IFNs. For example, IFN-γ has a 10-foldlower specific anti-viral activity than either IFN-α or IFN-β. On theother hand IFN-γ is 100-10,000 times more active as an immune systemmodulator than are the other classes of interferon, having potentphagocyte-activating effects not seen with other interferon types (Paceet al., 1985).

IFN-γ is a 20-25 kDa glycoprotein that exists as a homodimer insolution. Recombinant IFN-γ has an elimination half-life in thebloodstream after intravenous (iv) administration of 25-35 min and isessentially undetectable after 4 hours depending on the dose. Thesubcutaneous (sc) route generally results in a somewhat extendedhalf-life of 5-6 hours (see review by Younes and Amsden, 2002). Therecommended dosing schedule for IFN-γ is a sc injection of 50 μg/m²three times weekly. Relevant to this invention is the fact that IFN-γhas a short elimination half-life, whether given iv or sc, and thusrequires relatively frequent re-administration.

Several studies have demonstrated that continuous exposure to IFN-γenhances the protein's potency. Researchers have investigated thebenefits of continuous infusion of IFN-γ versus once dailyintraperitoneal injections in Leishmania donavani-infected mice. Dailydosing of IFN-γ did induce anti-microbial resistance, but these effectswere considerably enhanced by continuous administration of a comparabledose (47% reduction in liver parasite burden versus 9%) (Murray, 1990).In a similar study, mice were infected with Mycobacterium tuberculosis.Continuous delivery of IFN-γ via an external pump, prolonged survivallonger than did daily intramuscular IFN-γ injections (12 vs. 4 dayslonger than controls) (Flynn et al., 1993). These studies suggest asuperior therapeutic benefit from constant circulating levels of IFN-γ.Unfortunately, continuous infusion of IFN-γ is not practical for mostpatients. Therefore the need still exists for a long acting form ofIFN-γ.

Covalent modification of proteins with polyethylene glycol (PEG) hasproven to be a useful method to extend the circulating half-lives ofproteins in the body (Abuchowski et al., 1984; Meyers et al., 1991;Keating et al., 1993). Several PEGylated proteins are approved for usein humans or are in human clinical trials (Harris et al., 2003).Covalent attachment of PEG to a protein increases the protein'seffective size and reduces its rate of clearance from the body,presumably through interference with protein removal pathways, includingkidney glomerular filtration, proteolytic degradation as well as activeclearance via specific receptors.

Given the therapeutic value of long-acting forms of IFN-γ, as well asother therapeutic proteins and peptides, there is a continued need inthe art to provide new variants of IFN-γ that have improved stability,higher potency, greater solubility, longer circulating half-lives forless frequent dosing, and reduced antigenicity as compared to the parent(native) protein. In addition, since expression of recombinant proteinscontaining free cysteine residues has been problematic due to reactivityof the free sulfhydryl at physiological conditions, there also remains aneed in the art for improved, cost-effective methods for manufacturing(producing) such proteins that result in high yields of biologicallyactive product.

SUMMARY OF THE INVENTION

One embodiment of the invention relates to a method for refolding aninsoluble protein or peptide that lacks essential disulfides and thatcomprises at least one free cysteine residue, comprising the followingsteps: (a) causing a host cell to express a protein or peptide thatlacks essential disulfides and that comprises at least one free cysteineresidue in an insoluble or aggregated form; (b) lysing the host cell;(c) isolating the protein or peptide; (d) denaturing and reducing theprotein or peptide in a solution comprising both a denaturing agent anda reducing agent, wherein said reducing agent does not form a mixeddisulfide with the free cysteine in the protein or peptide, and whereinsaid reducing agent does not inactivate a thiol-reactive polyethyleneglycol (PEG) or does not interfere with modification of the protein by athiol-reactive PEG reagent; and (e) refolding the protein by reducingthe concentrations of the denaturing agent and reducing agents in thesolution of (d) to levels sufficient to allow the protein or peptide torenature into a soluble, biologically active form.

In one aspect, the step of lysing can be performed by chemical,enzymatic or physical means. In one aspect, the step of lysing compriseslysing the host cell in the presence of a denaturing agent. In anotheraspect, the step of lysing comprises lysing the host cell in thepresence of a denaturing agent and the reducing agent used in step (d).

In one aspect of the method, the denaturing agent is selected from thegroup consisting of: urea, guanidine and N-lauroyl sarcosine.

In one aspect of the method, the reducing agent in step (d) is areducing agent that does not contain a thiol moiety. In another aspect,the reducing agent in step (d) is a phosphine reductant. In one aspect,the reducing agent in step (d) is an alkyl phosphine. In one aspect, thealkyl phosphine can include, but is not limited to, a butyl phosphine, ahydroxypropyl phosphine, a cyanoethyl phosphine, and a carboxyethylphosphine. In one aspect, the alkyl phosphine can include, but is notlimited to: tri-n-butylphosphine (TBP), tris(hydroxypropyl)phosphine(THP), tris(2-cyanoethyl)phosphine (TCNP), andtris(2-carboxyethyl)phosphine (TCEP), or a combination thereof. In apreferred aspect, the alkyl phosphine is an ester of a carboxyethylphosphine. In another preferred aspect, the reducing agent isTris(2-carboxyethyl)phosphine-HCl (TCEP).

In one aspect of the method, step (e) occurs in the presence ofsufficient reducing agent to prevent the protein from forming disulfidebonds. In one aspect, step (e) of refolding comprises refolding theprotein in the presence of glycerol. In one aspect, step (e) ofrefolding comprises refolding the protein in the presence of anoxidizing agent selected from oxygen, iodine, hydrogen peroxide,dihydroascorbic acid, tetrathionate, or O-iodosobenzoate. In one aspect,step (e) of refolding comprises refolding the protein in the presence ofa metal ion. Such a metal ion can include, but is not limited to, Cu⁺⁺or Co⁺⁺.

The method of the invention can further include a step of isolating therefolded protein or peptide from other proteins and contaminants in therefold mixture. In one aspect, the protein is isolated from othercontaminants in the refold mixture by column chromatography. Forexample, in one aspect, the column chromatography buffers can contain areducing agent, wherein said reducing agent does not form a mixeddisulfide with the free cysteine in the protein or peptide, and whereinsaid reducing agent does not inactivate a thiol-reactive polyethyleneglycol (PEG) or does not interfere with modification of the protein by athiol-reactive PEG reagent. In one aspect, the reducing agent used inthe column is the same reducing agent used in step (d).

The method of the invention can also include a further step of exposingthe protein or peptide to a cysteine-reactive moiety to obtain acysteine-modified protein, wherein the cysteine-reactive moiety isattached to at least one added cysteine in said isolated protein orpeptide. In one aspect, the step of exposing is conducted in thepresence of a reducing agent. For example, in one aspect, the reducingagent is the same reducing agent used in step (d). Cysteine-reactivemoieties can include, but are not limited to, a polyethylene glycol, apolyvinyl pyrolidone, a carbohydrate, a dextran, a peptide, a lipid anda polysaccharide. In a preferred embodiment, the cysteine-reactivemoiety is a polyethylene glycol.

The proteins to be refolded using the method of the invention caninclude any protein or peptide that lacks essential disulfides and thatcomprises at least one free cysteine residue, including, but not limitedto a cysteine variant of interferon-gamma (IFN-γ), a cysteine variant ofinterleukin-11, or a cysteine variant of growth hormone. In one aspect,the protein is a cysteine variant of IFN-γ, and the amino acid Q1 isdeleted or substituted by a non-glutamine amino acid, and/or the aminoacid D2 is deleted or substituted by a non-aspartic acid amino acid.Peptides that can be refolded using the method of the invention include,but are not limited to, a cysteine variant of a bioactive peptideselected from: glucagon, glucagon-like peptide-1 (GLP-1), GLP-2, Gastricinhibitory peptide (GIP), PYY, exendin, ghrelin, gastrin, amylin, andoxyntomodulin.

Another embodiment of the invention relates to a cysteine variant ofinterferon-gamma (IFN-γ) (SEQ ID NO:1) isolated from step (e) of themethod of refolding of claim 1, wherein the cysteine variant of IFN-γdoes not contain mixed disulfides.

One embodiment of the invention relates to a method for refolding aninsoluble protein or peptide that lacks essential disulfides and thatcomprises at least one free cysteine residue, comprising the followingsteps: (a) causing a host cell to express a protein or peptide thatlacks essential disulfides and that comprises at least one free cysteineresidue in an insoluble or aggregated form; (b) lysing the host cell;(c) isolating the protein or peptide; (d) denaturing and reducing theprotein or peptide in a solution comprising both a denaturing agent anda reducing agent; (e) refolding the protein by reducing theconcentrations of the denaturing agent and reducing agents in thesolution of (d) to levels sufficient to allow the protein or peptide torenature into a soluble, biologically active form; (f) isolating theprotein from other contaminants in the refold mixture by columnchromatography, wherein the column chromatography buffers contain areducing agent, wherein said reducing agent does not form a mixeddisulfide with the free cysteine in the protein or peptide, and whereinsaid reducing agent does not inactivate a thiol-reactive polyethyleneglycol (PEG) or does not interfere with modification of the protein by athiol-reactive PEG reagent. In one aspect, the reducing agent in step(f) is TCEP.

Yet another embodiment of the invention relates to a cysteine variant ofinterferon-gamma (IFN-γ) (SEQ ID NO:1), wherein a cysteine residue issubstituted for at least one amino acid located in at least one regionof IFN-gamma selected from the group consisting of: the E-F loop and theregion following helix F. In one aspect, a cysteine residue issubstituted for an amino acid selected from: V99, V100, T101, D102,L103, P122, A123, A124, K125, T126, G127, K128, R129, K130, R131, S132,Q133, M134, L135, F136, R137, G138, R139, R140, A141, S143, or Q143. Inone aspect, the variant is modified with a cysteine reactive moiety,such as a polyethylene glycol. In one aspect, Q1 is deleted orsubstituted by a non-glutamine amino acid. In another aspect, D2 isdeleted or substituted by a non-aspartic acid amino acid.

Another embodiment of the invention relates to a method for treating adisease treatable with IFN-gamma, comprising administering to saidpatient a therapeutically effective amount of a cysteine variant ofIFN-gamma as described herein. Such a disease includes, but is notlimited to, chronic granulomatous disease, severe malignantosteopetrosis, or cancer.

Yet another embodiment of the invention relates to a cysteine analog ofglucagon-like peptide-1 (GLP-1) (SEQ ID NO:9), wherein a cysteineresidue is added to the peptide either by substitution for a nativeamino acid in GLP-1 or by insertion preceding the first amino acid ofGLP-1 or following the last amino acid of GLP-1. In one aspect, acysteine residue is substituted for at least one amino acid selectedfrom the group consisting of H1, A2, E3, G4, T5, F6, T7, S8, D9, V10,S11, S12, Y13, L13, E14, G15, Q16, A17, A18, K19, E20, F21, I22, A23,W24, L25, V26, K27, G28, R29, or G30. In another aspect, a cysteineresidue is inserted preceding the first amino acid of GLP-1 or followingthe last amino acid of GLP-1.

Another embodiment of the invention relates to a cysteine analog ofexendin (SEQ ID NO:11), wherein a cysteine residue is added to thepeptide either by substitution for a native amino acid in exendin or byinsertion preceding the first amino acid of exendin or following thelast amino acid of exendin. In one aspect, a cysteine residue issubstituted for an amino acid selected from the group consisting of H1,G2, E3, G4, T5, F6, T7, S8, D9, L10, S11, K12, Q13, M14, E15, E16, E17,A18, V19, R20, L21, F22, I23, E24, W25, L26, K27, N28, G29, G30, P31,S32, S33, G34, A35, P36, P37, and S38. In another aspect, a cysteineresidue is inserted preceding the first amino acid of exendin orfollowing the last amino acid of exendin.

Another embodiment of the invention relates to a cysteine analog ofamylin (SEQ ID NO:17), wherein a cysteine residue is added to thepeptide either by substitution for a native amino acid in amylin or byinsertion preceding the first amino acid of amylin or following the lastamino acid of amylin. In one aspect, a cysteine residue is substitutedfor an amino acid selected from the group consisting of K1, N3, T4, A5,T6, A8, T9, Q10, R11, L12, A13, N14, F15, L16, V17, H18, S19, S20, N21,N22, F23, G24, A25, I26, L27, S28, S29, T30, N31, V32, G33, S34, N35,T36, and Y37. In another aspect, a cysteine residue is insertedpreceding the first amino acid of amylin or following the last aminoacid of amylin.

Yet another embodiment of the invention relates to a cysteine analog ofglucagon (SEQ ID NO:8), wherein a cysteine residue is added to thepeptide either by substitution for a native amino acid in glucagon or byinsertion preceding the first amino acid of glucagon or following thelast amino acid of glucagon. In one aspect, a cysteine residue issubstituted for an amino acid selected from the group consisting of H1,S2, Q3, G4, T5, F6, T7, S8, D9, Y10, S11, K12, Y13, L14, D15, S16, R17,R18, A19, Q20, D21, F22, V23, Q24, W25, L26, M27, N28, and T29. Inanother aspect, a cysteine residue is inserted preceding the first aminoacid of glucagon or following the last amino acid of glucagon.

Another embodiment of the invention relates to a cysteine analog ofGLP-2 (SEQ ID NO:10), wherein a cysteine residue is added to the peptideeither by substitution for a native amino acid in GLP-2 or by insertionpreceding the first amino acid of GLP-2 or following the last amino acidof GLP-2. In one aspect, a cysteine residue is substituted for an aminoacid selected from the group consisting of H1, A2, D3, G4, S5, F6, S7,D8, E9, M10, N11, T12, I13, L14, D15, N16, L17, A18, A19, R20, D21, F22,I23, N24, W25, L26, I27, Q28, T29, K30, I31, T32, and D33. In anotheraspect, a cysteine residue is inserted preceding the first amino acid ofGLP-2 or following the last amino acid of GLP-2.

Yet another embodiment of the invention relates to a cysteine analog ofPeptide YY (PYY) (SEQ ID NO:12), wherein a cysteine residue is added tothe peptide either by substitution for a native amino acid in PYY or byinsertion preceding the first amino acid of PYY or following the lastamino acid of PYY. In one aspect, a cysteine residue is substituted foran amino acid selected from the group consisting of Y1, P2, 13, K4, P5,E6, A7, P8, G9, E10, D11, A12, S13, P14, E15, E16, L17, N18, R19, Y20,Y21, A22, S23, L24, R25, H26, Y27, L28, N29, L30, V31, T32, R33, Q34,R35, and Y36. In another aspect, a cysteine residue is insertedpreceding the first amino acid of PYY or following the last amino acidof PYY.

Another embodiment of the invention relates to a cysteine analog ofghrelin (SEQ ID NO:13), wherein a cysteine residue is added to thepeptide either by substitution for a native amino acid in ghrelin or byinsertion preceding the first amino acid of ghrelin or following thelast amino acid of ghrelin. In one aspect, a cysteine residue issubstituted for an amino acid selected from the group consisting of G1,S2, S3, F4, L5, S6, P7, E8, H9, Q10, R11, V12, Q13, Q14, R15, K16, E17,S18, K19, K20, P21, P22, A23, K24, L25, Q26, P27, and R28. In anotheraspect, a cysteine residue is inserted preceding the first amino acid ofghrelin or following the last amino acid of ghrelin. In one aspect, S3of ghrelin is acetylated.

Another embodiment of the invention relates to cysteine analog ofoxyntomodulin (SEQ ID NO:14), wherein a cysteine residue is added to thepeptide either by substitution for a native amino acid in oxyntomodulinor by insertion preceding the first amino acid of oxyntomodulin orfollowing the last amino acid of oxyntomodulin. In one aspect, acysteine residue is substituted for an amino acid selected from thegroup consisting of H1, S2, Q3, G4, T5, F6, T7, S8, D9, Y10, S11, K12,Y13, L14, D15, S16, R17, R18, A19, Q20, D21, F22, V23, Q24, W25, L26,M27, D28, T29, K30, R31, N32, K33, N34, N35, 136, and A37. In anotheraspect, a cysteine residue is inserted preceding the first amino acid ofoxyntomodulin or following the last amino acid of oxyntomodulin.

Another embodiment of the invention relates to a cysteine analog of GIP(SEQ ID NO:16), wherein a cysteine residue is added to the peptideeither by substitution for a native amino acid in GIP or by insertionpreceding the first amino acid of GIP or following the last amino acidof GIP. In one aspect, a cysteine residue is substituted for an aminoacid selected from the group consisting of Y1, A2, E3, G4, T5, F6, I7,S8, D9, Y10, S11, I12, A13, M14, D15, K16, I17, H18, Q19, Q20, D21, F22,V23, N24, W25, L26, L27, A28, Q29, K30, G31, K32, K33, N34, D35, W36,K37, H38, N39, I40, T41 and Q42. In another aspect, a cysteine residueis inserted preceding the first amino acid of GIP or following the lastamino acid of GIP.

In one aspect of any of the above-identified embodiments related tocysteine analogs, the added cysteine residue is modified with acysteine-reactive moiety. For example, the cysteine-reactive moiety caninclude, but is not limited to, a polyethylene glycol.

Another embodiment of the invention relates to a lysine analog of amylin(SEQ ID NO:17), wherein a lysine residue is added to the peptide eitherby substitution for a native amino acid in amylin or by insertionpreceding the first amino acid of amylin or following the last aminoacid of amylin. In one aspect, a lysine residue is substituted for anamino acid selected from the group consisting of N3, T4, A5, T6, A8, T9,Q10, R11, L12, A13, N14, F15, L16, V17, H18, S19, S20, N21, N22, F23,G24, A25, I26, L27, S28, S29, T30, N31, V32, G33, S34, N35, T36, andY37. In another aspect, a lysine residue is inserted preceding the firstamino acid of amylin or following the last amino acid of amylin. In oneaspect, K1 is changed to a non-lysine amino acid.

Yet another embodiment of the invention relates to a lysine analog ofglucagon (SEQ ID NO:8), wherein a lysine residue is added to the peptideeither by substitution for a native amino acid in glucagon or byinsertion preceding the first amino acid of glucagon or following thelast amino acid of glucagon. In one aspect, a lysine residue issubstituted for an amino acid selected from the group consisting of H1,S2, Q3, G4, T5, F6, T7, S8, D9, Y10, S11, Y13, L14, D15, S16, R17, R18,A19, Q20, D21, F22, V23, Q24, W25, L26, M27, N28, and T29. In anotheraspect, a lysine residue is inserted preceding the first amino acid ofglucagon or following the last amino acid of glucagon. In one aspect,K12 is changed to a non-lysine amino acid.

Another embodiment of the invention relates to a lysine analog ofglucagon-like peptide-1 (GLP-1) (SEQ ID NO:9), wherein a lysine residueis added to the peptide either by substitution for a native amino acidin GLP-1 or by insertion preceding the first amino acid of GLP-1 orfollowing the last amino acid of GLP-1. In one aspect, a lysine residueis substituted for at least one amino acid selected from the groupconsisting of H1, A2, E3, G4, T5, F6, T7, S8, D9, V10, S11, S12, Y13,L13, E14, G15, Q16, A17, A18, E20, F21, I22, A23, W24, L25, V26, G28,R29, and G30. In one aspect, a lysine residue is inserted preceding thefirst amino acid of GLP-1 or following the last amino acid of GLP-1. Inanother aspect, K20 is changed to a non-lysine amino acid. In oneaspect, K28 is changed to a non-lysine amino acid. In another aspect,K20 and K28 are changed to non-lysine amino acids, or K19 and K27 arechanged to non-lysine amino acids.

Yet another embodiment of the invention relates to a lysine analog ofGLP-2 (SEQ ID NO:10), wherein a lysine residue is added to the peptideeither by substitution for a native amino acid in GLP-2 or by insertionpreceding the first amino acid of GLP-2 or following the last amino acidof GLP-2. In one aspect, a lysine residue is substituted for an aminoacid selected from the group consisting of H1, A2, D3, G4, S5, F6, S7,D8, E9, M10, N11, T12, I13, L14, D15, N16, L17, A18, A19, R20, D21, F22,I23, N24, W25, L26, I27, Q28, T29, I31, T32, and D33. In one aspect, alysine residue is inserted preceding the first amino acid of GLP-2 orfollowing the last amino acid of GLP-2. In another aspect, K30 ischanged to a non-lysine amino acid.

Another embodiment of the invention relates to a lysine analog ofexendin (SEQ ID NO:11), wherein a lysine residue is added to the peptideeither by substitution for a native amino acid in exendin or byinsertion preceding the first amino acid of exendin or following thelast amino acid of exendin. In one aspect, a lysine residue issubstituted for an amino acid selected from the group consisting of H1,G2, E3, G4, T5, F6, T7, S8, D9, L10, S11, Q13, M14, E15, E16, E17, A18,V19, R20, L21, F22, I23, E24, W25, L26, N28, G29, G30, P31, S32, S33,G34, A35, P36, P37, and S38. In another aspect, a lysine residue isinserted preceding the first amino acid of exendin or following the lastamino acid of exendin. In one aspect, K12 is changed to a non-lysineamino acid. In another aspect, K27 is changed to a non-lysine aminoacid. In yet another aspect, K12 and K27 are changed to non-lysine aminoacids.

Another embodiment of the invention relates to a lysine analog ofoxyntomodulin (SEQ ID NO:14), wherein a lysine residue is added to thepeptide either by substitution for a native amino acid in oxyntomodulinor by insertion preceding the first amino acid of oxyntomodulin orfollowing the last amino acid of oxyntomodulin. In one aspect, a lysineresidue is substituted for an amino acid selected from the groupconsisting of H1, S2, Q3, G4, T5, F6, T7, S8, D9, Y10, S11, Y13, L14,D15, S16, R17, R18, A19, Q20, D21, F22, V23, Q24, W25, L26, M27, D28,T29, R31, N32, N34, N35, 136, and A37. In one aspect, a lysine residueis inserted preceding the first amino acid of oxyntomodulin or followingthe last amino acid of oxyntomodulin. In one aspect, an amino acidselected from K12, K30 and K33 is changed to a non-lysine amino acid. Inone aspect, two or more lysine residues selected from the groupconsisting of K12, K30 and K33 are changed to non-lysine amino acids. Inone aspect, K12, K30 and K33 are changed to non-lysine amino acids.

Another embodiment of the invention relates to a lysine analog ofghrelin (SEQ ID NO:13), wherein a lysine residue is added to the peptideeither by substitution for a native amino acid in ghrelin or byinsertion preceding the first amino acid of ghrelin or following thelast amino acid of ghrelin. In one aspect, a lysine residue issubstituted for an amino acid selected from the group consisting of G1,S2, S3, F4, L5, S6, P7, E8, H9, Q10, R11, V12, Q13, Q14, R15, E17, S18,P21, P22, A23, L25, Q26, P27, and R28. In one aspect, a lysine residueis inserted preceding the first amino acid of ghrelin or following thelast amino acid of ghrelin. In one aspect, S3 of ghrelin is acetylated.In another aspect, an amino acid selected from K16, K19, K20, and K24 ischanged to a non-lysine amino acid. In one aspect, two or more lysineresidues selected from the group consisting of K16, K19, K20 and K24 arechanged to non-lysine amino acids. In another aspect, K12, K19, K20 andK24 are changed to non-lysine amino acids.

Yet another embodiment of the invention relates to a lysine analog ofPeptide YY (PYY) (SEQ ID NO:12), wherein a lysine residue is added tothe peptide either by substitution for a native amino acid in PYY or byinsertion preceding the first amino acid of PYY or following the lastamino acid of PYY. In one aspect, a lysine residue is substituted for anamino acid selected from the group consisting of Y1, P2, I3, P5, E6, A7,P8, G9, E10, D11, A12, S13, P14, E15, E16, L17, N18, R19, Y20, Y21, A22,S23, L24, R25, H26, Y27, L28, N29, L30, V31, T32, R33, Q34, R35, andY36. In another aspect, a lysine residue is inserted preceding the firstamino acid of PYY or following the last amino acid of PYY. In anotheraspect, K4 is changed to a non-lysine amino acid.

Another embodiment of the invention relates to a lysine analog of GIP(SEQ ID NO:16), wherein a lysine residue is added to the peptide eitherby substitution for a native amino acid in GIP or by insertion precedingthe first amino acid of GIP or following the last amino acid of GIP. Inone aspect, a lysine residue is substituted for an amino acid selectedfrom the group consisting of Y1, A2, E3, G4, T5, F6, I7, S8, D9, Y10,S11, I12, A13, M14, D15, I17, H18, Q19, Q20, D21, F22, V23, N24, W25,L26, L27, A28, Q29, G31, N34, D35, W36, H38, N39, I40, T41 and Q42. Inone aspect, a lysine residue is inserted preceding the first amino acidof GIP or following the last amino acid of GIP. In another aspect, anamino acid selected from K16, K30, K32, K33, and K37 is changed to anon-lysine amino acid. In one aspect, two or more lysine residuesselected from the group consisting of K16, K30, K32, K33 and K37 arechanged to non-lysine amino acids. In another aspect, K16, K30, K32, K33and K37 are changed to non-lysine amino acids.

In any of the above-embodiments related to lysine analogs, in oneaspect, the added lysine residue is modified with an amine-reactivemoiety. For example, such an amine-reactive moiety can include, but isnot limited to, a polyethylene glycol.

DETAILED DESCRIPTION OF THE INVENTION

One embodiment of the invention describes novel polymer IFN-γ conjugatesthat have been created by the chemical coupling of polymers such aspolyethylene glycol moieties to IFN-γ. Based on the present inventors'work, these novel IFN-γ-based protein polymer conjugates will haveimproved stability, higher potency, greater solubility, longercirculating half-lives for less frequent dosing, and reducedantigenicity as compared to the parent (native or wild-type) protein.Methods of making and using the variants are encompassed by theinvention.

Additional embodiments of the invention related to novel analogs ofbioactive peptides that may be used to create longer acting versions ofthe peptides. The longer-acting peptides are useful because they can beadministered at lower doses and less frequently than the unmodifiedpeptides. Such bioactive peptides include, but are not limited to,glucagon, glucagon-like peptide-1 (GLP-1), GLP-2, Gastric inhibitorypeptide (GIP), PYY, exendin, ghrelin, gastrin, amylin, andoxyntomoldulin. Methods of making and using such peptides are alsoencompassed by the invention.

In one embodiment, the invention provides a novel method for producing,and particularly, refolding, the novel IFN-γ-based protein polymerconjugates of the invention, which can be extended to the production ofany protein or peptide that does not contain essential disulfides (i.e.disulfides that are essential for the proper folding and/or biologicalactivity of the protein), and that contains at least one free cysteineresidue.

Various aspects of the present invention are described in detail below,although particular examples are not intended to limit the scope of thepresent invention.

Method of Producing Proteins and Peptides According to the Invention

One embodiment of the present invention relates to a novel method torefold a protein or a peptide that does not contain essential disulfidesand that comprises at least one free cysteine. More particularly,cysteine residues in most proteins participate in disulfide bonds andare not available for derivatization without significant loss ofbioactivity. These disulfide bonds are referred to herein as “essential”disulfides, in that they are required for the proper folding and/or thebiological activity of the protein. However, some proteins and peptidescontain “non-essential” disulfides, i.e., disulfide bonds that are notrequired for the proper folding of the protein and/or are not requiredfor biological activity of the protein. In addition, some proteins andpeptides contain no native cysteine residues and thus contain no nativedisulfide bonds. In addition, some proteins naturally contain, or can bemodified to contain, one or more free cysteine residues that do notparticipate in disulfides. For example, through in vitro site-directedmutagenesis techniques, additional cysteine residues can be introducedat a specified site on a protein or peptide, e.g., by substitution orinsertion. Any free cysteine in a protein or peptide, including a freecysteine that occurs in the native (wild-type) protein, a free cysteinethat is created by deleting or substituting a non-cysteine residue forone cysteine in a non-essential disulfide, or a newly added “free”cysteine (e.g., as a result of substitution of a cysteine for anon-cysteine residue or the insertion of a cysteine into a protein orpeptide), can then serve as the site for the specific conjugation with athiol-reactive moiety (e.g., thiol-reactive polyethylene glycol). Thepresent invention provides a novel method of efficiently and effectivelyproducing such proteins and peptides in a cost-effective manner.

Accordingly, the present invention encompasses a wide variety ofrecombinant proteins, and cysteine variants of these proteins. Examplesof proteins that can be refolded using the method of the inventioninclude, but are not limited to, cysteine variants of: interferon-gamma(IFN-γ), interleukin-11 (IL-11), growth hormone, glucagon, glucagon-likepeptide-1 (GLP-1), GLP-2, Gastric inhibitory peptide (GIP), PYY,exendin, ghrelin, gastrin, amylin, oxyntomoldulin, and other proteinsand peptides that do not contain native cysteine residues. Any proteinor peptide that has at least one free cysteine (native or added bymodification of the protein or peptide) and does not otherwise containessential disulfides can be refolded using this method. Such proteinsinclude proteins that lack essential disulfides, but contain at leastone free cysteine that is not involved in a disulfide bond; proteinsthat lack essential disulfides, but contain non-essential disulfides,where the protein has been modified to delete one of the cysteines inthe non-essential disulfide pair (leaving one free cysteine); orproteins or peptides in which a free cysteine has been added byinserting it before the first amino acid of the protein or after thelast amino acid of the protein, or by inserting it between two of thenative amino acid residues, or by substituting a cysteine residue for anon-cysteine residue in the protein.

The method of the invention generally includes the steps of: (a) causinga host cell to express a protein or peptide that lacks essentialdisulfides and that comprises at least one free cysteine residue in aninsoluble or aggregated form; (b) lysing the host cell; (c) isolatingthe protein or peptide; (d) denaturing and reducing the protein orpeptide in a solution comprising both a denaturing agent and a reducingagent, wherein said reducing agent does not form a mixed disulfide withthe free cysteine in the protein or peptide, and wherein said reducingagent does not inactivate a thiol-reactive polyethylene glycol (PEG) ordoes not interfere with modification of the protein by a thiol-reactivePEG reagent; and (e) refolding the protein by reducing theconcentrations of the denaturing agent and reducing agents in thesolution of (d) to levels sufficient to allow the protein or peptide torenature into a soluble, biologically active form. If the user of themethod is provided with an isolated, insoluble or aggregated protein orpeptide that lacks essential disulfides and that comprises at least onefree cysteine residue, then the method can also begin with step (d),since steps (a) through (c) may have been performed independently. Inanother embodiment, the cell can be lysed directly into the solutioncomprising the denaturing agent and the reducing agent described above,rather than isolating the protein first as in step (c). Optionally, inany of the above-methods, the refolded, soluble protein can be isolatedfrom other proteins in the refold mixture. In a preferred embodiment,the refolded protein is isolated from other proteins in the refoldmixture by column chromatography. Preferably, the column chromatographybuffers used to isolate the refolded protein contain a reducing agentthat does not inactivate a thiol-reactive PEG reagent, or does notinterfere with modification of the protein by a thiol-reactive PEGreagent. In another embodiment, a reducing agent that does inactivate athiol-reactive PEG can be used to reduce the protein in step (d), butthe protein is purified subsequently using column buffers that contain areducing agent that does not inactivate a thiol-reactive PEG reagent, ordoes not interfere with modification of the protein by a thiol-reactivePEG reagent.

One advantage of the method of the invention, however, is that theproteins do not necessarily have to be purified away from the reducingagent used in step (d) prior to further modification with athiol-reactive polyethylene glycol, because the reducing agent does notinactivate PEG or interfere with modification of the protein by athiol-reactive PEG reagent. In methods described prior to the invention,a protein refolded using other thiol-reactive reducing agents (e.g.,DTT, cysteine) that do inactivate thiol-reactive PEGs or interfere withmodification of a protein with a thiol-reactive PEG, would have to bedialyzed to remove the reducing agent prior to PEGylation. Therefore,PEGylation of proteins refolded by the method of the invention is moreefficient and removes a step in the PEGylation process (and thus is morecost effective). In addition, the use of a reducing agent specified inthe present method (does not form a mixed disulfide with the freecysteine in the protein or peptide, and does not inactivate athiol-reactive polyethylene glycol (PEG) or interfere with modificationof a protein with a thiol-reactive PEG), the resulting product isqualitatively better than with certain reducing agents (e.g., cysteine),because the resulting refolded protein is mostly non-disulfide bondedand monomeric (or homodimeric in the case of interferon gamma), whereasother reducing agents can produce more disulfide-linked dimers,tetramers, or multimers, or undesired disulfide-linked aggregates. Themonomeric form, or in the case of certain proteins (e.g., interferongamma), a homodimeric form, is the preferred commercial product, and somethods that enhance the ability to produce and recover these preferredforms is of great value.

Accordingly, the use of a reducing agent such as the exemplary TCEP inthe method of the invention both improves the quality of the refoldedproduct (i.e., more non-disulfide bonded product) and allows efficientPEGylation of the refolded product because the agent does not have to beremoved prior to PEGylation. Indeed, if desired, such a reducing agentcould be added as a reducing agent in the PEGylation process or orpurification steps prior to PEGylation to facilitate the PEGylationreaction.

The reducing agent for use in the denaturing and reducing step (d) (andin some embodiments, in subsequent steps) is preferably a phosphinereductant and, more preferably, an alkyl phosphine. Exemplary alkylphosphines for use in the methods of the present invention include, butare not limited to, alkyl phosphines such as tri-n-butylphosphine (TBP),hydroxypropyl phosphines such as tris(hydroxypropyl)phosphine (THP),cyanoethyl phosphines such as tris(2-cyanoethyl)phosphine (TCNP),carboxyethyl phosphines such as tris(2-carboxyethyl)phosphine (TCEP) andany combination of these reductants. Additionally, esters of thecarboxyethyl phosphines such as monomethyl-, dimethyl- ortrimethyl-esters of tris(2-carboxyethyl)phosphine (TCEP) or combinationsthereof may be used in the denaturing and reducing reactions of theinventive processes. The selection of the appropriate phosphinereductant(s) for use in the methods of the present invention will dependupon the chemical characteristics of the isolated protein or peptide tobe refolded using the methodology described herein. For example, aprotein isolated and stabilized at an acidic pH less than about 5.5having one or more free cysteine residues may require a phosphinereducing agent having a stronger reducing reactivity at a lower pH, suchas the trimethylester of TCEP, while retaining the biochemicalcharacteristics of acting in concert with a denaturing agent in a singledenaturing and reducing step, and limited or absent inhibitory effect onthe PEGylation reaction, such that it does not need to be removed bydialysis or other methods prior to PEGylation. Similarly, a proteinisolated and stabilized at a more basic pH of about 8.0 having one ormore free cysteine residues may require a phosphine reducing agenthaving a stronger reducing reactivity at a higher pH, such as TCEP.Preferably, the reducing agent for use in the protein denaturing andreducing reactions of the present invention is an alkyl phosphine, morepreferably the reducing agent is an ester derivative of TCEP, and mostpreferably the reducing agent is TCEP.

In another embodiment, the reducing agent for use in the denaturing andreducing steps, and/or in subsequent reducing steps of the method of theinvention, is a reducing agent that does not contain a sulfur moiety.

As identified above, the first step in the method of the invention is tocause a host cell to express a protein having a free cysteine residue inan insoluble or aggregated form. Suitable host cells can be prokaryoticor eukaryotic. Examples of appropriate host cells that can be used toexpress recombinant proteins include bacteria, yeast, insect andmammalian cells. Bacteria cells are particularly useful, especially E.coli. Methods of causing a host cell to express a protein are well knownin the art and examples are provided herein.

As used herein, the term “protein having a free cysteine residue” meansany natural or recombinant protein or peptide that contains 2N+1cysteine residues, where N can be 0 or any integer, and any natural orrecombinant protein or peptide that contain 2N cysteines, where two ormore of the cysteines do not normally participate in a disulfide bond.Thus, the methods of the present invention are useful in enhancing theexpression, recovery and purification of any protein or peptide having afree cysteine, particularly cysteine added variant recombinant proteins(referred to herein as “cysteine muteins” or “cysteine variants”) havingone or more free cysteines. As previously discussed, the invention isprimarily intended to be used for proteins that do not contain anessential disulfide (two cysteines that form a disulfide bond that isessential to the ability of the protein to fold and/or have biologicalactivity). Although the expression, recovery and purification of anatural protein having a free cysteine expressed by its natural hostcell can be enhanced by the methods of the present invention, thedescription herein predominantly refers to recombinant proteins forillustrative purposes only. In addition, the proteins can be derivedfrom any animal species including human, companion animals and farmanimals. The proteins also can be derived from plant species ormicrobes.

In one step of the method, after the protein is expressed by the cell,the host cell is lysed. Cell lysis can occur prior to, or coincidentwith, the solubilization procedures described herein. Cell lysis can beaccomplished by, for example, mechanical sheer such as a French pressurecell, enzymatic digestion, sonication, homogenization, glass beadvortexing, detergent treatment, organic solvents, freeze thaw, grindingwith alumina or sand, treatment with a denaturing agent as definedbelow, and the like (Bollag et al., 1996). Optionally, the cells can belysed in the presence of a denaturing agent and a disulfide reducingagent, preferably those in the solution used to solubilize the protein.Optionally, insoluble or aggregated material can be separated fromsoluble proteins prior to the solubilization step by various methodssuch as centrifugation, filtration (including ultrafiltration),precipitation, floculation, or settling.

Next the insoluble or aggregated material (or whole cells without priorlysis) is rendered soluble or monomeric by exposing the insoluble oraggregated material (or whole cells without prior lysis) to a solutioncomprising both a denaturing agent and a disulfide reducing agent thatdoes not form a mixed disulfide with the free cysteine in the protein orpeptide, and does not inactivate a thiol-reactive polyethylene glycol(PEG).

Useful denaturing agents include urea, guandine, arginine, sodiumthiocyanate, extremes in pH (dilute acids or bases), detergents (SDS,sarkosyl), salts (chlorides, nitrates, thiocyanates, cetylmethylammoniumsalts, trichloroacetates), chemical derivatization (sulfitolysis,reaction with citraconic anhydride), solvents(2-amino-2-methyl-1-propanol or other alcohols, DMSO, DMF) or stronganion exchange resins such as Q-Sepharose. Useful concentrations of ureaare 1-8 M, with 5-8 M being preferred concentrations. Usefulconcentrations of guanidine are 1-8 M, with 4-8 M being preferredconcentrations.

Useful disulfide reducing agents are any reducing agents that do notform a mixed disulfide with the free cysteine in the protein or peptide,and that do not inactivate a thiol-reactive polyethylene glycol (PEG),or interfere with modification of the protein with a thiol-reactive PEG.In one embodiment, such a reducing agent is a phosphine reductant and,more preferably, an alkyl phosphine. In one embodiment, an alkylphosphine includes, but is not limited to, an alkyl phosphine such astri-n-butylphosphine (TBP), a hydroxypropyl phosphine such astris(hydroxypropyl)phosphine (THP), a cyanoethyl phosphine such astris(2-cyanoethyl)phosphine (TCNP), a carboxyethyl phosphine such astris(2-carboxyethyl)phosphine (TCEP) and any combination of thesereductants. In one embodiment, the reducing ageint is an ester of thecarboxyethyl phosphines such as monomethyl-, dimethyl- ortrimethyl-esters of tris(2-carboxyethyl)phosphine (TCEP) or combinationsthereof. In one preferred embodiment, the reducing agent is an esterderivative of TCEP, and most preferably the reducing agent is TCEP. Inone embodiment, the reducing agent is a reducing agent that does notcontain a sulfur moiety.

These reducing agents can be used in the range of 0.5 to 200 mM, with1-50 mM being preferred concentrations. Notably, it is not necessary touse a cysteine blocking agent in the method of the invention, becausethe method is used to refold proteins that do not contain (lack)essential disulfides, and so there is no need to block the freecysteines in the protein. Accordingly, in one aspect, the reducing agentis not a cysteine blocking agent. Preferably, the pH of thedenaturation/reduction mixture is between pH 6 and pH 10.

The next step in the method of the invention is to refold the protein toobtain the protein's native conformation so that it can be furthermodified in biologically active form by a polymer, such as polyethyleneglycol. Refolding is achieved by reducing the concentrations of thedenaturing agent and reducing agent to levels sufficient to allow theprotein to renature into a soluble, biologically active form. This canbe achieved through dialysis, dilution, gel filtration, precipitation ofthe protein, or by immobilization on a resin followed by buffer washes.Preferably, a reagent or combination of reagents are chosen that resultin refolding of the protein, while maintaining the protein in a reducedstate so that the free cysteine(s) in the protein can be derivatized.Optionally, the reducing agent used in the refold step can be added tothe dialysis buffer to keep the protein reduced during the renaturationprocess. Useful concentrations of the reducing agent in the dialysisbuffer are 1 μM to 100 mM, with 0.1 mM to 10 mM being a preferredconcentration and 1 mM being the most preferred concentration.Optionally, a metal ion such as copper (Cu⁺⁺) or cobalt (Co⁺⁺) can beadded to the refold mixture to promote protein oxidation. Usefulconcentrations of metal ions in the refold mixture are 1 μM to 1 mM,with 40 μM being a preferred concentration. Preferably, the pH of therefold mixture is between pH 6 and pH 10.

Optionally, the refolded, soluble protein containing a free cysteineresidue is recovered and isolated from other proteins in the solublefraction of the refold mixture. Such recovery and purification methodsare known or readily determined by those skilled in the art, including,for example, centrifugation, filtration, dialysis, chromatography,including size exclusion, ion-exchange, hydrophobic interaction andaffinity chromatography procedures and the like. A suitable method forthe recovery and purification of a desired protein will depend, in part,on the properties of the protein and the intended use. In a preferredembodiment the refolded protein is isolated from other proteins in therefold mixture by column chromatography. Preferably, the columnchromatography buffers used to isolate the refolded protein contain areducing agent that does not inactivate a thiol-reactive PEG reagent, ordoes not interfere with modification of the protein by a thiol-reactivePEG reagent. Useful concentrations of the reducing agent in the columnchromatography buffers are 1 μM to 100 mM, with 0.1 mM to 10 mM being apreferred concentration and 1 mM being the most preferred concentration.If a reducing agent that does inactivate a thiol-reactive PEG is used toreduce the protein in step (d) above, the protein may be isolated fromother proteins in the refold mixture using column chromatography buffersthat contain a reducing agent that does not inactivate a thiol-reactivePEG reagent, or does not interfere with modification of the protein by athiol-reactive PEG reagent.

The purified proteins obtained according to these methods can be furtherprocessed if desired, and indeed, the method of the invention isparticularly useful for subsequent modification of the protein orpeptide at the free cysteine residue with various cysteine-reactivemoieties. For example, the proteins can be PEGylated at the freecysteine residue with various cysteine-reactive PEG reagents, andsubsequently purified as monoPEGylated proteins. The term“monoPEGylated” is defined to mean a protein modified by covalentattachment of a single PEG molecule to the protein. Any method known tothose skilled in the art can be used to purify the PEGylated proteinfrom unmodified protein and unreacted PEG reagents, including, forexample, the methods described in the Examples below, and inPCT/US98/14497 and PCT/US00/00931. Examples of other usefulcysteine-reactive moieties are cysteine-reactive dextrans,cysteine-reactive carbohydrates and cysteine-reactivepoly(N-vinylpyrrolidone)s.

One exemplary method of refolding a protein according to the presentinvention is described in detail in Example 3, wherein the reducingagent is TCEP. This example is for purposes of illustration only, andthe invention is not limited to the details presented in this example.

The present invention further relates to purified, monoPEGylated orPEGylated protein variants produced by the methods disclosed herein thatare not only biologically active, but also retain high specific activityin protein-dependent mammalian cell proliferation assays.

PEG Conjugation of Proteins According to the Invention

PEGs are commercially available in several sizes (2-40 kDa) and shapes(linear and branched), allowing the circulating half-lives ofPEG-modified proteins to be tailored for individual indications throughthe use of different PEGs. PEGylation increases a protein's effectivemolecular weight more than would be expected based on the molecularweight of the PEG moiety due to the water of hydration associated withthe PEG group. For example, attachment of a single 5 kDa PEG to a 36 kDaprotein increases the effective molecular weight of the complex togreater than 100 kDa, as measured by size-exclusion chromatography (Fee,2003). When administered by subcutaneous injection, PEGylated proteinsare slowly absorbed from the injection site, thus avoiding the serum“spikes” seen after subcutaneous injection of an unmodified protein.This “controlled release” of the PEGylated protein results in a moreconstant serum level, thus prolonging or increasing the drug'spharmacologic activity while minimizing the side effects typically seenwith fluctuations in the drug concentrations. Other documented in vivobenefits of PEG modification include an increase in protein solubility,enhanced stability (possibly due to protection from proteases) and adecrease in immunogenicity (Keating et al., 1993).

A PEG moiety can be attached to the N-terminal amino acid, a cysteineresidue (either native or non-native), lysines (either native ornon-native) or other reactive native or non-native amino acids in theprotein's primary sequence. A non-native amino acid is defined as anamino acid that is not normally located at that position in the protein,an amino acid analog that is not commonly seen in native proteins, or anamino acid or amino acid analog that has been chemically modified toallow conjugation with a polymer such as polyethylene glycol. Althoughspecific examples are provided herein with respect to the PEGylation ofIFN-γ, it is to be understood that any protein or peptide having a freecysteine and that is produced by the method of the invention can bePEGylated using the methods described below.

Amine Reactive PEG Reagents for Modification of IFN-γ

The most common route for PEG conjugation of proteins is to use a PEGwith a functional group that reacts with lysines and/or the N-terminalamino acid group. The literature describes more than a dozen suchprocedures (see reviews by Hooftman et al., 1996; Delgato et al., 1992;and Zalipsky, 1995). Examples of amine-reactive PEGs include PEGdichlorotriazine, PEG tresylate, PEG succinimidyl carbonate, PEGbenzotriazole carbonate, PEG p-nitrophenyl carbonate, PEGcarbonylimidazole, PEG succinimidyl succinate, PEG propionaldehyde, PEGacetaldehyde, and PEG hydroxysuccinimide.

Dimeric IFN-γ has 42 potential sites (two N-termini and 40 lysines) forconjugation with an amine reactive PEG. Multiple attachments may occurif the protein is exposed to an excess amount of PEGylation reagent.Preferably, the IFN-γ PEG conjugate would have 1-5 PEGs attached to thedimeric protein, more preferred would be 1-3 attachments, and mostpreferred 1-2 attachments. Conditions can be adjusted to limit thenumber of attachments or the site of attachments. The number ofattachments can be titrated by varying the molar ratios of thePEG:Protein. Preferred ratios can be determined experimentally. A secondmethod for varying the number of attachments is by modifying thereaction conditions. For example, the coupling can be preferentiallydirected to the alpha-terminus of a protein chain by performing thereaction at a pH lower than 7 and preferably below 6.5. Above pH 8, theepsilon-NH3 groups found on the lysines will be most reactive. (Morpurgoand Veronese, 2004). A third approach to controlling the number orlocation of the PEG conjugates is to conduct the PEGylation in thepresence of a substrate, reversible inhibitor or binding protein so thatthe active site or receptor binding site is protected during coupling. Afourth approach to controlling the number of attachments involves usinga larger PEG. For example, when interferon-alpha is modified with asmall linear polymer, up to 11 positional isomers are present in thefinal mixture. When interferon-alpha is modified with a larger 40 kDabranched PEG, only four main positional isomers are present in themono-PEGylated protein (Monkarsh et al., 1997, Foser et al. 2003,Baillon et al. 2003). A fifth method to control the number of attachedPEGs is to use column chromatography procedures (ion exchange, sizeexclusion or hydrophobic interaction) to purify a IFN-γ conjugatecontaining the desired number of PEG molecules from a more complexIFN-γ-PEG mixture. Amine PEGylation of IFN-γ has been described by Kitaet al. (1990). Amino acids 1 and lysine 129 or lysine 131 were modifiedby amine-reactive PEGs.

PEG-Protein Conjugates Using Cysteine-Reactive PEGs

A second method for PEGylating proteins covalently attaches PEG tocysteine residues using thiol-reactive PEGs. A number of highlyspecific, thiol-reactive PEGs with different reactive groups (e.g.,PEG-ortho-pyridyl-disulfide, PEG-maleimide, PEG-vinylsulfone andPEG-iodoacetamide), different size PEGs (2-40 kDa), different shapedPEGs (linear or branched) and different end group (hydroxyl, carboxylicacid, methoxy or other alkoxy group) are commercially available. Theconjugates are hydrolytically stable and the PEGylation reactions can beperformed at neutral pH.

As discussed above, cysteine residues in most proteins participate indisulfide bonds (essential disulfides) and are not available forderivatization. However, any free cysteine in a protein or peptide,including a free cysteine that occurs in the native (wild-type) protein,a free cysteine that is created by deleting or substituting anon-cysteine residue for one cysteine in a non-essential disulfide, or anewly added “free” cysteine (e.g., as a result of substitution of acysteine for a non-cysteine residue or the insertion of a cysteine intoa protein or peptide), will serve as the site for the specificattachment of a PEG molecule, thus avoiding the product heterogeneitythat is often seen with amine PEGylation reactions. The added cysteinemust be exposed on the protein's surface and be accessible forPEGylation for this method to be successful. If the chosen site isnon-essential (e.g. for proper folding or biological activity of theprotein), then the PEGylated protein will display wild type (normal) ornear wild type in vitro bioactivity.

A free thiol group can also be introduced into the primary amino acidsequence of a protein by chemical modification of lysine. One suchexample involves treatment of the protein with Traut's reagent(2-Iminothiolane hydrochloride). Alternatively the protein can betreated with reagents such as N-succinimidyl S-acetylthioacetate (SATA)or N-succinimidyl S-acetylthiopropionate (SATP) that introduce aprotected sulfhydryl which can be deprotected prior to exposure to athiol reactive PEG. Alternatively, a “free” cysteine can be introducedby deleting or mutating a native cysteine (that normally forms adisulfide bond) to another amino acid such as a serine or alanine sothat an odd number of cysteines are present in the protein's primarysequence.

It is also possible to genetically introduce two or more additionalcysteines that are not able to disulfide bond with each other. In thiscase, two PEGs can be specifically attached to the protein.Alternatively, a native disulfide bond can be reduced, resulting in twofree cysteine residues that are available for thiol specific PEGylation.Free thiol groups can also be introduced by chemical conjugation of apeptide to a protein where the peptide contains a free cysteine group ora cysteine group modified with a reversible thiol-blocking agent.

Carboxyl-Reactive PEGs for PEGylation

PEG-hydrazide can be used to PEGylate the carboxyl groups of amino acidsin presence of N,N′-dicyclohexylcarbodiimide (DCC), or in presence of awater soluble coupling agent such asN-(-3dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC). Thecarboxyl groups of a protein when activated with EDC at an acidic pH (pH4.5-5) react readily with PEG-hydrazide, whereas amino groups of theprotein are protonated and unreactive. Dimeric IFN-γ has 20 asparticacids (D2, D21, D24, D41, D61, D62, D76, D90, D91 and D102) and 18glutamic acids (E7, E9, E38, E39, E71, E75, E93, E112 and E119).

PEGylation of Unnatural Amino Acids

Similar to the genetically engineered cysteine mutations forsite-specific PEGylation, researchers have reported the specificincorporation of unnatural amino acids into proteins expressed in yeast,in E. Coli and in mammalian cells (Deiters et al., 2004). Specificallypara-azidophenylalanine was substituted into a protein at certain sitesdetermined by the positioning of the amber codon. The reactive group onthe amino acid analog was used in a mild [3+2] cycloaddition reactionwith an alkyne derivatized PEG reagent to allow for site-specificconjugation.

Arginine-Reactive PEGs for PEGylation

Another approach is the attachment of the PEG moiety on the arginineside chain using PEG-1-3-dioxo compounds such as PEG-phenylglioxate. Thedisadvantages of this approach include long reaction times and limitedspecificity since other amino acids such as histidine and lysine, may bemodified as well. Dimeric IFN-γ has 16 arginines (R42, R89, R107, R129,R131, R137, R139, and R140).

Hydroxyl-Reactive PEGs for PEGylation

PEG-isocyanate can be used to attach a PEG to a hydroxy group via astable urethane linkage. The disadvantage of this approach is lack ofspecificity since it is also capable of reacting with amines. Thus, thisreagent is more commonly used in PEGylation reactions involvingpolysaccharides or non-peptide drugs.

Oxidized Carbohydrate-Reactive PEGs for PEGylation

Oxidation of the carbohydrate residues or N-terminal serine or threonineis an alternative method for a site-specific PEGylation. Carbohydrateside chains can be oxidized with enzymes or chemically with sodiumperiodate to generate reactive aldehyde groups. These sites can bereacted with either PEG-hydrizide or PEG-amine to produce a reversibleSchiff's base. These linkages are then reduced with sodiumcyanoborohydride to a more stable alkyl hydrazide or in the case of theSchiff's base, a secondary amine. Multiple attachment sites aregenerated by this method but the PEG is localized on the carbohydratechain rather than on the protein.

A similar approach takes advantage of an N-terminal serine or threonine.These amino acid residues can be converted by periodate oxidation to aglyoxylyl derivative that will also react with PEG-hydrazide orPEG-amine. IFN-γ variants in which a serine or threonine residue isadded preceding Q1 or substituted for Q1 are contemplated by thisinvention.

Enzymatically Catalyzed PEGylation Reagents

Another approach for PEGylation of proteins uses transglutaminase tomodify glutamine residues so that they become reactive with alkylaminederivatives of PEG (Sato 2002). Dimeric IFN-γ has 18 glutamines (Q1,Q45, Q47, Q64, Q67, Q106, Q115, Q133, and Q143).

N-Terminal PEGylation for Proteins Containing an N-Terminal Cysteine orN-Terminal Histidine

IFN-γ can be modified to contain an N-terminal cysteine or N-terminalhistidine which can be specifically PEGylated on the N-terminal amineusing an o-pyridylthioester PEG reagent (Nektar). The reagent initiallyreacts with the amino acid side chain followed by a chemicalrearrangement that results in an amide linkage between the PEG and theamino group of the N-terminal amino acid.

Multifunctional PEGs

While a majority of the PEGylated proteins currently on the market haveone or more PEGs per protein, it is also possible to construct proteinconjugates with two or more proteins attached to one PEG moiety.Difunctional and heterofunctional PEGs are commercially available andcan be used to covalently link two or more IFN-γ variants.

Bioactive Peptides of the Invention, and Polymer Conjugates Thereof

One embodiment of the present invention relates to PEGylated bioactivepeptides, and particularly, PEGylated cysteine variants of bioactivepeptides, and to any homologues thereof, or truncated forms thereof. Inone aspect, the bioactive peptide is a peptide wherein the nativeprotein contains no essential disulfides, and wherein the peptidecontains, or has been modified to contain, at least one free cysteine.In one embodiment, such a bioactive peptide is produced by a method ofthe invention. In another aspect, the invention relates to bioactivepeptides that are PEGylated variants of a peptide selected from thegroup consisting of glucagon, glucagon-like peptide-1 (GLP-1), GLP-2,Gastric inhibitory peptide (GIP), PYY, exendin, ghrelin, gastrin,amylin, and oxyntomoldulin.

Another embodiment of the present invention relates to a compositioncomprising any one or more of the above-described polymer conjugated ornon-conjugated variants of the bioactive peptides, including cysteinevariants, homologues thereof, or truncated forms thereof, and apharmaceutically acceptable carrier.

Yet another embodiment of the present invention relates to a cysteinevariant of any of glucagon, glucagon-like peptide-1 (GLP-1), GLP-2,Gastric inhibitory peptide (GIP), PYY, exendin, ghrelin, gastrin,amylin, and oxyntomoldulin, such as those described in detail in theExamples, or a homologue or truncated form thereof, wherein a cysteineresidue is substituted for at least one amino acid in the peptide.Preferred sites for cysteine substitution are described in Examples11-19.

In one aspect of this embodiment, the substituted or inserted cysteineresidue is modified with a cysteine-reactive moiety. In another aspect,the substituted or inserted cysteine residue is modified withpolyethylene glycol.

Another embodiment of the present invention relates to a compositioncomprising any one or more (at least 2, 3, 4, 5, 6, 7, 8, 9 or 10) ofthe above-described variants and a pharmaceutically acceptable carrier.

Many bioactive peptides have been described, including glucagon,glucagon-like peptide-1 (GLP-1), GLP-2, Gastric inhibitory peptide(GIP), PYY, exendin, ghrelin, gastrin, amylin, and oxyntomoldulin. Thesepeptides typically are 10-40 amino acids in length and most do notcontain cysteine residues. Methods to develop longer acting forms ofthese peptides are desired. In the following Examples we provide novelanalogs of these peptides that may be used to create longer actingversions of the peptides. The longer-acting peptides are useful becausethey can be administered lat lower doses and less frequently than theunmodified peptides. Preferred analogs are ones in which a cysteineresidue has been introduced into the peptide sequence either bysubstitution for a native amino acid or by insertion preceding the firstamino acid or following the last amino acid of the peptide. Cysteineresidues also can be inserted between two adjacent amino acids in thepeptide chain. The added cysteine residue can be modified with acysteine reactive moiety such as a cysteine-reactive polymer. Usefulcysteine reactive polymers include, but are not limited tocysteine-reactive polyethylene glycols (PEGs), dextrans, sugars, andstarches. PEGs can be linear or branched PEGs. Other usefulcysteine-reactive moieties for modifying the peptides include but arenot limited to cysteine-reactive lipids, labels, dyes, lipids,chromphores, and radionucleotides. Preferred reactive groups on thecysteine-reactive moieties include maleimide, vinylsulfone andiodoacetamide groups. The methods described herein are particularlyuseful for modifying peptides that do not contain a native cysteineresidue (s) with a cysteine-reactive moiety such as a cysteine-reactivePEG. These methods also can be used to modify peptides that do containone or more native cysteine residues. These methods also are useful forcreating longer-acting antagonist analogs of peptides.

Alternatively, the peptides can be modified with amine-reactive moietiessuch as amine-reactive polymers, labels, dyes, lipids, chromphores, andradionucleotides. Useful amine-reactive polymers include, but are notlimited to, amine-reactive PEGs, dextrans, sugars and starches.Typically amine-reactive moieties attach to lysine residues or theN-terminal amino acid. Labeling of the peptides can be made morespecific for lysine residues or the N-terminus by substitutingnon-lysine amino acids, preferably arginine, for one or more lysineresidues in the protein. Additionally, new lysine residues can beengineered into the peptides to create new sites for modification of thepeptides. New lysine residues can be added to the protein bysubstitution for a non-lysine residue or by insertion preceding thefirst amino acid of the peptide or following the last amino acid of thepeptide. Lysine residues also can be inserted between two adjacent aminoacids in the peptide chain. These methods also are useful for creatinglonger-acting agonist and antagonist analogs of peptides.

Methods for modifying a protein with a variety of polymers, including atcysteine and lysine residues, have been described above and areencompassed by this embodiment of the invention.

As used herein the term analog comprises a variety of modifications thatcan be made to a protein. Such changes may include amino acid variants,deletions, additions, insertions, fusion proteins, and covalent andnon-covalent modifications.

Also encompassed by the invention is to the use of any variant of abioactive peptide described herein, including any polymer-variantconjugate (e.g., a PEGylated bioactive peptide, including amonoPEGylated bioactive peptide) in a therapeutic method or in thepreparation of a medicament, such as for the treatment of a disease orcondition in which the use of the native bioactive peptide would beuseful. Such diseases and conditions are described for multiplebioactive peptide variants of the invention in the Examples.

IFN-γ Conjugates of the Invention

One embodiment of the present invention relates to PEGylated IFN-γvariants, and particularly, a PEGylated IFN-γ cysteine variant, and toany homologues thereof, or truncated forms thereof. In one aspect ofthis embodiment, the IFN-γ variant is a variant of a native IFN-γprotein comprising an amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2,with SEQ ID NO:1 being preferred.

Another embodiment of the present invention relates to a compositioncomprising any one or more of the above-described PEGylated IFN-γvariants, homologues thereof, or truncated forms thereof, and apharmaceutically acceptable carrier.

Yet another embodiment of the present invention relates to a cysteinevariant of IFN-γ (SEQ ID NO:1) or a homologue or truncated form thereof,wherein a cysteine residue is substituted for at least one amino acidlocated in at least one region of IFN-γ.

In one aspect of this embodiment, the substituted or inserted cysteineresidue is modified with a cysteine-reactive moiety. In another aspect,the substituted or inserted cysteine residue is modified withpolyethylene glycol.

Another embodiment of the present invention relates to a compositioncomprising any one or more (at least 2, 3, 4, 5, 6, 7, 8, 9 or 10) ofthe above-described variants and a pharmaceutically acceptable carrier.

Native IFN-γ is a homodimeric N-glycosylated protein with 143 aminoacids, no cysteine residues (i.e., no natural disulfides) and anisoelectric point of about 8.7. The amino acid sequence of the maturehuman IFN-γ protein is represented herein by SEQ ID NO:1, and the aminoacid sequence of the mature murine IFN-γ protein is represented hereinby SEQ ID NO:2. Amino acid variants and truncated forms of IFN-γ havebeen described (Van Dan Haze et al., 2005). When expressed in E. coli,the protein may contain an N-terminal methionine not found in the nativeprotein.

IFN-γ is enriched in basic amino acids with 40 lysines and 16 argininesper dimer. There are two potential glycosylation sites at N25 and N97.The absence of carbohydrate moieties in E. coli-derived recombinantIFN-γ does not affect its biological activity but it does have an effecton both its physicochemical and pharmacokinetic properties (Arakawa etal., 1985). Unlike the other IFN family members, IFN-γ is extremely acidsensitive. Between pH 3.5 and 4.5, the protein loses structuralintegrity and rapidly aggregates. At pH 2.3, an acid-labile bond isbroken with subsequent loss of all anti-viral activity (Mulkerrin andWetzel, 1989). IFN-γ is also heat sensitive and undergoes irreversiblethermal denaturation and aggregation in solution at temperatures greaterthan 40° C. (Beldarrain et al., 1999).

The X-ray crystal structure of recombinant human IFN-γ has beendetermined for both the protein itself (Ealick et al., 1991, Science252: 698-702) and in complex with the IFN-γ receptor alpha subunit(IFN-γRα) (Walter et al., 1995). IFN-γ is primarily alpha helical (˜62%)with no beta sheets. IFN-γ contains six alpha helical regions (helicesA-F) that are joined by short unstructured sequences or loops. Ealick etal., supra, identifies the helical and non-helical regions of humangamma interferon (FIG. 3 of Ealick et al.), wherein the positions of thehelical regions and the intervening (loop) regions, as well as theregion preceding Helix A and following Helix E, are located at thefollowing positions in the human gamma interferon amino acid sequence(SEQ ID NO:1): amino acids 4-15 (helix A), amino acids 28-36 (helix B),amino acids 41-62 (helix C), amino acids 68-82 (helix D), amino acids87-98 (helix E) and amino acids 104-121 (helix F). The region precedinghelix A accordingly encompasses amino acids 1-3, the A-B loopencompasses amino acids 16-27, the B-C loop encompasses amino acids37-40, the C-D loop encompasses amino acids 63-67, the D-E loopencompasses amino acids 83-86, the E-F loop encompasses amino acids99-103, and the region following helix F encompasses amino acids122-143.

IFN-γ exists as a dimer in solution that is stabilized by theintertwining of the helices across the subunit interface with multipleintersubunit interactions. The first four helices (A-D) from one subunitform a cleft that accommodates helix F of the other subunit. There arenumerous hydrophobic contacts among all of the helices, but most arebetween the C and D helices. The C helix is the most hydrophobic helixand is essentially buried in the core of the dimer. Overall, the IFN-γdimer has a “V”-like conformation with the amino and carboxyl regions ofeach monomer being juxtaposed to one another.

Preferred Sites for Thiol-Specific PEGylation of IFN-γ

In general, a cysteine substitution can be made for any amino acid thatis 1) located on the surface of IFN-γ, 2) not required for thestructural integrity of IFN-γ and 3) not required for receptor binding.Non-essential amino acids can be identified by performingcysteine-scanning mutagenesis on the target protein and measuringeffects on biological activity. Cysteine-scanning mutagenesis entailsadding or substituting cysteine residues for individual amino acids inthe polypeptide chain and determining the effect of the cysteinesubstitution on biological activity. Cysteine scanning mutagenesis issimilar to alanine-scanning mutagenesis (Cunningham et al., 1991),except that target amino acids are individually replaced with cysteinerather than alanine residues.

Glycosylation sites and glycosylation recognition sequences areattractive sites for attaching PEG molecules because these sites aresurface exposed and the natural protein can tolerate bulky sugar groupsat these positions. The human IFN-γ sequence contains two potentialN-linked glycosylation sites at asparagine 25 and asparagine 97.Asparagine 25 is located at the C-terminal end of the A-B loop.Aspargine 97 is located at the C-terminal end of Helix E. Tang et al.(1996) described the PEGylation of an IFN-γ variant in which asparagine97 was mutated to a cysteine residue. The N- and C-termini regions ofIFN-γ are also possible sites for site-directed PEGylation, includingadding a cysteine that precedes the first amino acid or follows the lastamino acid of IFN-γ. Cysteine insertions between two adjacent aminoacids within the primary amino acid sequence of IFN-γ are alsoreasonable sites for thiol specific PEGylation if the biologicalactivity or protein's conformation is not significantly affected by thecysteine insertions. Preferred sites for cysteine substitutions includethe region preceding helix A (Q1, D2, P3), the A-B loop region (N16,A17, G18, H19, S20, D21, V22, A23, D24, N25, G26), the B-C loop region(K37, E38, E39, S40), the C-D loop region (D63, Q64, S65, I66, Q67), theD-E loop region (N83, S84, N85, K86), the E-F loop region (N97, Y98,S99, V100, T101, D102, L103) and the region following Helix F (P122,A123, A124, K125, T126, G127, K128, R129, K130, R131, S132, Q133, M134,L135, F136, R137, G138, R139, R140, A141, S143, Q143). Cysteine residuesalso can be added preceding the protein's first amino acid or followingthe last amino acid. These IFN-γ cysteine variants can be incorporatedinto the native IFN-γ sequence or in variants in which Q1, D2, or Q1 andD2 are deleted or changed to non-glutamic or non-aspartic acids,respectively. Possible substitutions include amino acids such asalanine, arginine, aspartic acid (for Q1 only), asparagine, glutamicacid, glutamine (for D2 only), glycine, histidine, isoleucine, leucine,lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, or valine).

Therapeutic Uses for Long Acting IFN-γ proteins

Another embodiment of the invention relates to the use of any IFN-γvariant described herein, including any polymer IFN-γ variant conjugate(e.g., a PEGylated, including a monoPEGylated IFN-γ) in a therapeuticmethod or in the preparation of a medicament, such as for the treatmentof a disease or condition in which the use of native IFN-γ would beuseful.

A long acting form of IFN-γ would be useful for the treatment of anumber of immunological, viral, and neoplastic diseases, includingdiseases currently treated with IFN-γ. Recombinant IFN-γ is currentlyapproved by the FDA for reducing the frequency and severity of seriousinfections associated with chronic granulomatous disease (CGD) and fordelaying time to disease progression in patients with severe malignantosteopetrosis. CGD is a group of rare, inherited disorders of the immunesystem that are caused by defects in the phagocytes. These defects leavepatients vulnerable to severe reoccurring bacterial and fungalinfections along with chronic inflammatory conditions. Malignantosteopetrosis is an inherited disorder, characterized by an osteoclastdefect leading to bone overgrowth and deficient phagocyte oxidativemetabolism. This life-threatening disorder leads to blindness, deafnessby six months of age and an increase in susceptibility to infection.Treatment with IFN-γ has proven to be effective in delaying diseaseprogression from a median time of 65 days after birth for the controlgroup to 165 days for the treatment group (Key et al., 1995).

A long acting form of IFN-γ is useful for treating Idiopathic PulmonaryFibrosis (IPF), which is a disorder that is characterized by progressivescarring or fibrosis of the lungs, which ultimately leads to death.Another potential indication for a long acting form of IFN-γ is cancerand in particular, ovarian cancer where current treatment withchemotherapy alone has a five-year survival rate of only 44%. In invitro and in vivo studies, IFN-γ has been shown to be directly toxic tocancer cells, including ovarian cancer cells, neuroblastoma cancercells, and Ewings Sarcoma cancer cells, and to stimulate the body'simmune system to enhance the removal of cancer cells (see review by Wallet al., 2003). In vitro and in vivo studies have shown that IFN gammamay be useful as an anti-cancer agent alone and in combination withproteins that cause cell death such as TRAIL and TRAIL receptor ligandagonists (Yang et al., 2003; Merchant et al., 2004). IFN gamma inducesexpression of cell death proteases such as caspase-8 in many tumor cellsand may be useful in combination with other anti-cancer compounds thatrequire or use the caspase-8 cell death pathway to kill tumor cells. IFNgamma also may be useful as an anti-cancer agent in combination withchemotherapeutic agents that induce TRAIL receptor [either TrailReceptor 1 (TR1) or Trail Receptor 2 (TR2) or both) expression on tumorcells. IFN gamma can inhibit proliferation and survival of cancer cellsdirectly, or by inhibiting their metastasis to other tissues and organs.A long acting form of IFN-γ may be useful for treating liver fibrosis,asthma, and lymphomas, and IFN-γ also may prove to be an effectivetreatment against biological warfare agents. Preclinical data hasconfirmed IFN-γ's ability to protect against both small pox andtularaemia in animal models (Liu, 2003; Anthony et al., 1989). A longacting form of IFN-γ may be useful in the treatment of small pox,anthrax, plague, botulism, tularaemia, and hemorrhagic fevers.

A long acting form of IFN-γ can also be used alone or synergistically incombination with other anti-fungal, anti-viral, anti-bacterial oranti-tumor agents. For example, IFN-c′ and IFN-γ, while functionallyrelated, bind to different cell surface receptors (See review by Lau etal., 2002). In in vitro cells-based assays and in vivo animal studies,combinations of IFN-α and -γ show enhanced anti-viral and anti-tumoractivities than either drug alone (Larkin et al. 2003; Fleischmann etal., 1998; Yanai et al., 2001). Anti-fungal agents have been evaluatedin conjunction with IFN-γ and shown to be effective in previouslyunresponsive patients (Summers et al., 2005).

A long acting form of IFN-γ or composition comprising the same of thepresent invention is administered to an animal in a manner effective todeliver the composition to a target cell, a target tissue, orsystemically to the animal, whereby provision of a therapeutic benefitis achieved as a result of the administration of the IFN-γ orcomposition. Suitable administration protocols include any in vivo or exvivo administration protocol. According to the present invention,suitable methods of administering a composition of the present inventionto a patient include any route of in vivo administration that issuitable for delivering the composition into a patient. The preferredroutes of administration will be apparent to those of skill in the art,depending on the type of condition to be prevented or treated and/or thetarget cell population.

Compositions and Therapeutic Methods of the Invention

Any of the proteins or peptides described herein or produced by themethods of the invention, including long acting (e.g., PEGylated) formsof the proteins or peptides, can be used in various compositions andtherapeutic methods. Generally, the proteins and peptides of theinvention can be used in any method, including any therapeutic method,that is useful with the native (unmodified, wild-type) protein orpeptide.

Cysteine muteins and PEGylated proteins and peptides of the presentinvention are preferably administered in a composition. Compositions caninclude a cysteine mutein (cysteine variant) of the invention and anyother suitable pharmaceutically acceptable carrier, as well as, in someaspects, additional components that may be useful in the treatment of agive disease or condition. According to the present invention, a“pharmaceutically acceptable carrier” includes pharmaceuticallyacceptable excipients and/or pharmaceutically acceptable deliveryvehicles, which are suitable for use in administration of thecomposition to a suitable in vitro, ex vivo or in vivo site. A suitablein vitro, in vivo or ex vivo site is preferably any site where thecysteine mutein will provide a detectable effect as compared to in theabsence of the mutein, and includes a disease site or a site of celltypes to be contacted with the mutein. Preferred pharmaceuticallyacceptable carriers are capable of maintaining the mutein of the presentinvention in a form that, upon arrival of the mutein at the cell targetin a culture or in patient, the mutein is capable of interacting withits target (e.g., platelets or progenitor cells thereof).

Suitable excipients of the present invention include excipients orformularies that transport or help transport, but do not specificallytarget a composition to a cell or area (also referred to herein asnon-targeting carriers). Examples of pharmaceutically acceptableexcipients include, but are not limited to water, phosphate bufferedsaline, Ringer's solution, dextrose solution, serum-containingsolutions, Hank's solution, other aqueous physiologically balancedsolutions, oils, esters and glycols. Aqueous carriers can containsuitable auxiliary substances required to approximate the physiologicalconditions of the recipient, for example, by enhancing chemicalstability and isotonicity. Compositions of the present invention can besterilized by conventional methods and/or lyophilized.

One type of pharmaceutically acceptable carrier includes a controlledrelease formulation that is capable of slowly releasing a composition ofthe present invention into a patient or culture. As used herein, acontrolled release formulation comprises a cysteine mutein of thepresent invention in a controlled release vehicle. Suitable controlledrelease vehicles include, but are not limited to, biocompatiblepolymers, other polymeric matrices, capsules, microcapsules,microparticles, bolus preparations, osmotic pumps, diffusion devices,liposomes, lipospheres, and transdermal delivery systems. Other carriersof the present invention include liquids that, upon administration to apatient, form a solid or a gel in situ. Preferred carriers are alsobiodegradable (i.e., bioerodible). In the event that a cysteine muteinof the invention is administered as a recombinant nucleic acid moleculeencoding the cysteine mutein (e.g., gene therapy or geneticimmunization), suitable carriers include, but are not limited toliposomes, viral vectors or other carriers, including ribozymes, goldparticles, poly-L-lysine/DNA-molecular conjugates, and artificialchromosomes. Natural lipid-containing carriers include cells andcellular membranes. Artificial lipid-containing carriers includeliposomes and micelles.

A carrier of the present invention can be modified to target to aparticular site in a patient, thereby targeting and making use of acompound of the present invention at that site. A pharmaceuticallyacceptable carrier which is capable of targeting can also be referred toherein as a “delivery vehicle” or “targeting carrier”. Suitablemodifications include manipulating the chemical formula of the lipidportion of the delivery vehicle and/or introducing into the vehicle atargeting agent capable of specifically targeting a delivery vehicle toa preferred site or target site, for example, a preferred cell type. A“target site” refers to a site in a patient to which one desires todeliver a composition. Suitable targeting compounds include ligandscapable of selectively (i.e., specifically) binding another molecule ata particular site. Examples of such ligands include antibodies,antigens, receptors and receptor ligands. Manipulating the chemicalformula of the lipid portion of the delivery vehicle can modulate theextracellular or intracellular targeting of the delivery vehicle. Forexample, a chemical can be added to the lipid formula of a liposome thatalters the charge of the lipid bilayer of the liposome so that theliposome fuses with particular cells having particular chargecharacteristics.

One delivery vehicle of the present invention is a liposome. A liposomeis capable of remaining stable in an animal for a sufficient amount oftime to deliver a nucleic acid molecule or protein described in thepresent invention to a preferred site in the animal. A liposome,according to the present invention, comprises a lipid composition thatis capable of delivering a nucleic acid molecule or protein to aparticular, or selected, site in a patient. A liposome according to thepresent invention comprises a lipid composition that is capable offusing with the plasma membrane of the targeted cell to deliver anucleic acid molecule or protein into a cell. Suitable liposomes for usewith the present invention include any liposome. Preferred liposomes ofthe present invention include those liposomes commonly used in, forexample, gene delivery methods known to those of skill in the art. Morepreferred liposomes comprise liposomes having a polycationic lipidcomposition and/or liposomes having a cholesterol backbone conjugated topolyethylene glycol. Complexing a liposome with a nucleic acid moleculeor protein of the present invention can be achieved using methodsstandard in the art.

Another type of delivery vehicle, when the cysteine mutein isadministered as a nucleic acid encoding the mutein, comprises a viralvector. A viral vector includes an isolated nucleic acid molecule, inwhich the nucleic acid molecules are packaged in a viral coat thatallows entrance of DNA into a cell. A number of viral vectors can beused, including, but not limited to, those based on alphaviruses,poxviruses, adenoviruses, herpesviruses, lentiviruses, adeno-associatedviruses and retroviruses.

According to the present invention, an effective administration protocol(i.e., administering a therapeutic composition in an effective manner)comprises suitable dose parameters and modes of administration thatresult in the desired effect in the patient (e.g., stimulation ofplatelet production), preferably so that the patient is protected fromthe disease (e.g., by disease prevention or by alleviating one or moresymptoms of ongoing disease). Effective dose parameters can bedetermined using methods standard in the art for a particular disease.Such methods include, for example, determination of survival rates, sideeffects (i.e., toxicity) and progression or regression of disease.

In accordance with the present invention, a suitable single dose size isa dose that results in the desired therapeutic effect in the patient,depending on the cysteine mutein that is administered, or in theamelioration of at least one symptom of a condition in the patient, whenadministered one or more times over a suitable time period. Doses canvary depending upon the disease being treated. One of skill in the artcan readily determine appropriate single dose sizes for a given patientbased on the size of a patient and the route of administration.

In one aspect of the invention, a suitable single dose of a therapeuticcomposition of the present invention is an amount that, whenadministered by any route of administration, provides a therapeuticeffect in the patient as described above, as compared to a patient whichhas not been administered with the therapeutic composition of thepresent invention (i.e., a control patient), as compared to the patientprior to administration of the composition, or as compared to a standardestablished for the particular disease, patient type and composition.

In one aspect of the invention an appropriate single dose of a cysteinemutein of the present invention is at least about 0.01 micrograms per kgof the animal to which the mutein is administered, and in other aspects,at least about 0.1 micrograms/kg, at least about 0.2 micrograms/kg, atleast about 0.5 micrograms/kg, at least about 1 micrograms/kg, at leastabout 5 micrograms/kg, at least about 10 micrograms/kg, at least about25 micrograms/kg, at least about 50 micrograms/kg, at least about 75micrograms/kg, at least about 100 micrograms/kg, at least about 200micrograms/kg, at least about 300 micrograms/kg, at least about 400micrograms/kg, at least about 500 micrograms/kg, at least about 750micrograms/kg, at least about 1 mg/kg, or at least about 5 mg/kg.

As discussed above, a therapeutic composition of the present inventionis administered to a patient in a manner effective to deliver thecomposition to a cell, a tissue, and/or systemically to the patient,whereby the desired result is achieved as a result of the administrationof the composition. Suitable administration protocols include any invivo or ex vivo administration protocol. The preferred routes ofadministration will be apparent to those of skill in the art, dependingon the type of condition to be prevented or treated; whether thecomposition is nucleic acid based or protein based; and/or the targetcell/tissue. For proteins or nucleic acid molecules, preferred methodsof in vivo administration include, but are not limited to, intravenousadministration, intraperitoneal administration, intramuscularadministration, intranodal administration, intracoronary administration,intraarterial administration (e.g., into a carotid artery), subcutaneousadministration, transdermal delivery, intratracheal administration,subcutaneous administration, intraarticular administration,intraventricular administration, inhalation (e.g., aerosol),intracranial, intraspinal, intraocular, intranasal, oral, bronchial,rectal, topical, vaginal, urethral, pulmonary administration,impregnation of a catheter, and direct injection into a tissue. Routesuseful for deliver to mucosal tissues include, bronchial, intradermal,intramuscular, intranasal, other inhalatory, rectal, subcutaneous,topical, transdermal, vaginal and urethral routes. Combinations ofroutes of delivery can be used and in some instances, may enhance thetherapeutic effects of the composition. Particularly preferred routes ofdelivery include subcutaneous and intravenous delivery.

Ex vivo administration refers to performing part of the regulatory stepoutside of the patient, such as administering a composition of thepresent invention to a population of cells removed from a patient underconditions such that the composition contacts and/or enters the cell,and returning the cells to the patient. Ex vivo methods are particularlysuitable when the target cell type can easily be removed from andreturned to the patient.

Many of the above-described routes of administration, includingintravenous, intraperitoneal, intradermal, and intramuscularadministrations can be performed using methods standard in the art.Aerosol (inhalation) delivery can also be performed using methodsstandard in the art (see, for example, Stribling et al., Proc. Nall.Acad. Sci. USA 189:11277-11281, 1992, which is incorporated herein byreference in its entirety). Oral delivery can be performed by complexinga therapeutic composition of the present invention to a carrier capableof withstanding degradation by digestive enzymes in the gut of ananimal. Examples of such carriers include plastic capsules or tabletssuch as those known in the art.

One method of local administration is by direct injection. Directinjection techniques are particularly useful for administering acomposition to a cell or tissue that is accessible by surgery, andparticularly, on or near the surface of the body. Administration of acomposition locally within the area of a target cell refers to injectingthe composition centimeters and preferably, millimeters from the targetcell or tissue.

Various methods of administration and delivery vehicles disclosed hereinhave been shown to be effective for delivery of a nucleic acid moleculeto a target cell, whereby the nucleic acid molecule transfected the celland was expressed. In many studies, successful delivery and expressionof a heterologous gene was achieved in preferred cell types and/or usingpreferred delivery vehicles and routes of administration of the presentinvention.

In the method of the present invention, compositions can be administeredto any animal and preferably, to any member of the Vertebrate class,Mammalia, including, without limitation, primates, rodents, livestockand domestic pets. Livestock include mammals to be consumed or thatproduce useful products (e.g., sheep for wool production). Preferredmammals to protect include humans, dogs, cats, mice, rats, sheep,cattle, horses and pigs, with humans being particularly preferred.

General Definitions

As used herein, reference to an isolated protein or polypeptide in thepresent invention, including an IFN-γ protein described particularlyherein, includes full-length proteins, fusion proteins, or any fragment(truncated form) or homologue of such a protein. Such a protein caninclude, but is not limited to, purified proteins, recombinantlyproduced proteins, membrane bound proteins, proteins complexed withlipids, soluble proteins and isolated proteins associated with otherproteins. More specifically, an isolated protein according to thepresent invention, is a protein (including a polypeptide or peptide)that has been removed from its natural milieu (i.e., that has beensubject to human manipulation) and can include purified proteins,partially purified proteins, recombinantly produced proteins, andsynthetically produced proteins, for example. As such, “isolated” doesnot reflect the extent to which the protein has been purified.Preferably, an isolated protein of the present invention is producedrecombinantly. In addition, and again by way of example, a “human IFN-γprotein” or a protein “derived from” a human IFN-γ protein refers to aIFN-γ protein (generally including a homologue of a naturally occurringIFN-γ protein) from a human (Homo sapiens) or to a IFN-γ protein thathas been otherwise produced from the knowledge of the structure (e.g.,sequence) and perhaps the function of a naturally occurring IFN-γprotein from Homo sapiens. In other words, a human IFN-γ proteinincludes any IFN-γ protein that has substantially similar structure andfunction of a naturally occurring IFN-γ protein from Homo sapiens orthat is a biologically active (i.e., has biological activity) homologueof a naturally occurring IFN-γ protein from Homo sapiens as described indetail herein. As such, a human IFN-γ protein can include purified,partially purified, recombinant, mutated/modified and syntheticproteins. According to the present invention, the terms “modification”and “mutation” can be used interchangeably, particularly with regard tothe modifications/mutations to the amino acid sequence of protein (ornucleic acid sequences) described herein. An isolated protein useful asan antagonist or agonist according to the present invention can beisolated from its natural source, produced recombinantly or producedsynthetically.

As used herein, the term “homologue” is used to refer to a protein orpeptide which differs from a naturally occurring protein or peptide(i.e., the “prototype” or “wild-type” protein) by modifications,including minor modifications, to the naturally occurring protein orpeptide, but which maintains the basic protein and side chain structureof the naturally occurring form. Such changes include, but are notlimited to: changes in one or a few (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9 or10) amino acid side chains; changes one or a few amino acids, includingdeletions (e.g., a truncated form of the protein or peptide) insertionsand/or substitutions; changes in stereochemistry of one or a few atoms;and/or minor derivatizations, including but not limited to: methylation,glycosylation, phosphorylation, acetylation, myristoylation,prenylation, palmitation, amidation and/or addition ofglycosylphosphatidyl inositol. A homologue can have either enhanced,decreased, or substantially similar properties as compared to thenaturally occurring protein or peptide. A homologue can include anagonist of a protein or an antagonist of a protein. A cysteine variantof IFN-γ is a homologue of IFN-γ, by way of example.

Homologues can be the result of natural allelic variation or naturalmutation. A naturally occurring allelic variant of a nucleic acidencoding a protein is a gene that occurs at essentially the same locus(or loci) in the genome as the gene which encodes such protein, butwhich, due to natural variations caused by, for example, mutation orrecombination, has a similar but not identical sequence. Allelicvariants typically encode proteins having similar activity to that ofthe protein encoded by the gene to which they are being compared. Oneclass of allelic variants can encode the same protein but have differentnucleic acid sequences due to the degeneracy of the genetic code.Allelic variants can also comprise alterations in the 5′ or 3′untranslated regions of the gene (e.g., in regulatory control regions).Allelic variants are well known to those skilled in the art.

Homologues can be produced using techniques known in the art for theproduction of proteins including, but not limited to, directmodifications to the isolated, naturally occurring protein, directprotein synthesis, or modifications to the nucleic acid sequenceencoding the protein using, for example, classic or recombinant DNAtechniques to effect random or targeted mutagenesis.

In one embodiment, a homologue of a given protein comprises, consistsessentially of, or consists of, an amino acid sequence that is at leastabout 45%, or at least about 50%, or at least about 55%, or at leastabout 60%, or at least about 65%, or at least about 70%, or at leastabout 75%, or at least about 80%, or at least about 85%, or at leastabout 90%, or at least about 95% identical, or at least about 95%identical, or at least about 96% identical, or at least about 97%identical, or at least about 98% identical, or at least about 99%identical (or any percent identity between 45% and 99%, in whole integerincrements), to the amino acid sequence of the reference protein. In oneembodiment, the homologue comprises, consists essentially of, orconsists of, an amino acid sequence that is less than 100% identical,less than about 99% identical, less than about 98% identical, less thanabout 97% identical, less than about 96% identical, less than about 95%identical, and so on, in increments of 1%, to less than about 70%identical to the naturally occurring amino acid sequence of thereference protein.

As used herein, unless otherwise specified, reference to a percent (%)identity refers to an evaluation of homology which is performed using:(1) a BLAST 2.0 Basic BLAST homology search using blastp for amino acidsearches and blastn for nucleic acid searches with standard defaultparameters, wherein the query sequence is filtered for low complexityregions by default (described in Altschul, S. F., Madden, T. L.,Schääffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J.(1997) “Gapped BLAST and PSI-BLAST: a new generation of protein databasesearch programs.” Nucleic Acids Res. 25:3389-3402, incorporated hereinby reference in its entirety); (2) a BLAST 2 alignment (using theparameters described below); (3) and/or PSI-BLAST with the standarddefault parameters (Position-Specific Iterated BLAST. It is noted thatdue to some differences in the standard parameters between BLAST 2.0Basic BLAST and BLAST 2, two specific sequences might be recognized ashaving significant homology using the BLAST 2 program, whereas a searchperformed in BLAST 2.0 Basic BLAST using one of the sequences as thequery sequence may not identify the second sequence in the top matches.In addition, PSI-BLAST provides an automated, easy-to-use version of a“profile” search, which is a sensitive way to look for sequencehomologues. The program first performs a gapped BLAST database search.The PSI-BLAST program uses the information from any significantalignments returned to construct a position-specific score matrix, whichreplaces the query sequence for the next round of database searching.Therefore, it is to be understood that percent identity can bedetermined by using any one of these programs.

Two specific sequences can be aligned to one another using BLAST 2sequence as described in Tatusova and Madden, (1999), “Blast 2sequences—a new tool for comparing protein and nucleotide sequences”,FEMS Microbiol Lett. 174:247-250, incorporated herein by reference inits entirety. BLAST 2 sequence alignment is performed in blastp orblastn using the BLAST 2.0 algorithm to perform a Gapped BLAST search(BLAST 2.0) between the two sequences allowing for the introduction ofgaps (deletions and insertions) in the resulting alignment. For purposesof clarity herein, a BLAST 2 sequence alignment is performed using thestandard default parameters as follows.

For blastn, using 0 BLOSUM62 matrix:Reward for match=1Penalty for mismatch=−2Open gap (5) and extension gap (2) penaltiesgap_x_dropoff (50) expect (10) word size (11) filter (on)For blastp, using 0 BLOSUM62 matrix:Open gap (11) and extension gap (1) penaltiesgap x_dropoff (50) expect (10) word size (3) filter (on).

According to the present invention, an isolated IFN-γ protein, includinga biologically active homologue or fragment thereof, has at least onecharacteristic of biological activity of activity the wild-type, ornaturally occurring IFN-γ protein (which can vary depending on whetherthe homologue or fragment is an agonist or antagonist of the protein, orwhether an agonist or antagonist mimetic of the protein is described).In general, the biological activity or biological action of a proteinrefers to any function(s) exhibited or performed by the protein that isascribed to the naturally occurring form of the protein as measured orobserved in vivo (i.e., in the natural physiological environment of theprotein) or in vitro (i.e., under laboratory conditions). Modifications,activities or interactions which result in a decrease in proteinexpression or a decrease in the activity of the protein, can be referredto as inactivation (complete or partial), down-regulation, reducedaction, or decreased action or activity of a protein. Similarly,modifications, activities or interactions which result in an increase inprotein expression or an increase in the activity of the protein, can bereferred to as amplification, overproduction, activation, enhancement,up-regulation or increased action of a protein. The biological activityof an IFN-γ protein according to the invention can be measured orevaluated using any assay for the biological activity of the protein asknown in the art. Such assays are known in the art, and assays for IFN-γactivity are described in the Examples.

In accordance with the present invention, an isolated polynucleotide(also referred to as an isolated nucleic acid molecule) is a nucleicacid molecule that has been removed from its natural milieu (e.g., thathas been subject to human manipulation), its natural milieu being thegenome or chromosome in which the nucleic acid molecule is found innature. As such, “isolated” does not necessarily reflect the extent towhich the nucleic acid molecule has been purified, but indicates thatthe molecule does not include an entire genome or an entire chromosomein which the nucleic acid molecule is found in nature. A polynucleotideuseful in the present invention can include a portion of a nucleic acidsequence (sense or non-sense strand) that is suitable for use as ahybridization probe or PCR primer for the identification of afull-length gene (or portion thereof), or to encode a protein orfragment (truncated form) or homologue thereof. An isolated nucleic acidmolecule that includes a gene is not a fragment of a chromosome thatincludes such gene, but rather includes the coding region and regulatoryregions associated with the gene, but no additional genes naturallyfound on the same chromosome. An isolated nucleic acid molecule can alsoinclude a specified nucleic acid sequence flanked by (i.e., at the 5′and/or the 3′ end of the sequence) additional nucleic acids that do notnormally flank the specified nucleic acid sequence in nature (i.e.,heterologous sequences). Isolated nucleic acid molecule can include DNA,RNA (e.g., mRNA), or derivatives of either DNA or RNA (e.g., cDNA).Although the phrase “nucleic acid molecule” primarily refers to thephysical nucleic acid molecule and the phrase “nucleic acid sequence”primarily refers to the sequence of nucleotides on the nucleic acidmolecule, the two phrases can be used interchangeably, especially withrespect to a nucleic acid molecule, or a nucleic acid sequence, beingcapable of encoding a protein. Preferably, an isolated nucleic acidmolecule of the present invention is produced using recombinant DNAtechnology (e.g., polymerase chain reaction (PCR) amplification,cloning) or chemical synthesis.

The minimum size of a nucleic acid molecule or polynucleotide of thepresent invention is a size sufficient to encode a protein having adesired biological activity, or sufficient to form a probe oroligonucleotide primer that is capable of forming a stable hybrid withthe complementary sequence of a nucleic acid molecule encoding thenatural protein (e.g., under moderate, high or very high stringencyconditions). If the polynucleotide is an oligonucleotide probe orprimer, the size of the polynucleotide can be dependent on nucleic acidcomposition and percent homology or identity between the nucleic acidmolecule and a complementary sequence as well as upon hybridizationconditions per se (e.g., temperature, salt concentration, and formamideconcentration). The minimum size of a polynucleotide that is used as anoligonucleotide probe or primer is at least about 5 nucleotides inlength, and preferably ranges from about 5 to about 50 or about 500nucleotides or greater, including any length in between, in whole numberincrements (i.e., 5, 6, 7, 8, 9, 10, . . . 33, 34, . . . 256, 257, . . .500). There is no limit, other than a practical limit, on the maximalsize of a nucleic acid molecule of the present invention, in that thenucleic acid molecule can include a portion of a protein-encodingsequence or a nucleic acid sequence encoding a full-length protein.

As used herein, stringent hybridization conditions refer to standardhybridization conditions under which nucleic acid molecules are used toidentify similar nucleic acid molecules. Such standard conditions aredisclosed, for example, in Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Labs Press, 1989. Sambrook et al.,ibid., is incorporated by reference herein in its entirety (seespecifically, pages 9.31-9.62). In addition, formulae to calculate theappropriate hybridization and wash conditions to achieve hybridizationpermitting varying degrees of mismatch of nucleotides are disclosed, forexample, in Meinkoth et al., 1984, Anal. Biochem. 138, 267-284; Meinkothet al., ibid., is incorporated by reference herein in its entirety.

More particularly, moderate stringency hybridization and washingconditions, as referred to herein, refer to conditions which permitisolation of nucleic acid molecules having at least about 70% nucleicacid sequence identity with the nucleic acid molecule being used toprobe in the hybridization reaction (i.e., conditions permitting about30% or less mismatch of nucleotides). High stringency hybridization andwashing conditions, as referred to herein, refer to conditions whichpermit isolation of nucleic acid molecules having at least about 80%nucleic acid sequence identity with the nucleic acid molecule being usedto probe in the hybridization reaction (i.e., conditions permittingabout 20% or less mismatch of nucleotides). Very high stringencyhybridization and washing conditions, as referred to herein, refer toconditions which permit isolation of nucleic acid molecules having atleast about 90% nucleic acid sequence identity with the nucleic acidmolecule being used to probe in the hybridization reaction (i.e.,conditions permitting about 10% or less mismatch of nucleotides). Asdiscussed above, one of skill in the art can use the formulae inMeinkoth et al., ibid. to calculate the appropriate hybridization andwash conditions to achieve these particular levels of nucleotidemismatch. Such conditions will vary, depending on whether DNA:RNA orDNA:DNA hybrids are being formed. Calculated melting temperatures forDNA:DNA hybrids are 10° C. less than for DNA:RNA hybrids. In particularembodiments, stringent hybridization conditions for DNA:DNA hybridsinclude hybridization at an ionic strength of 6×SSC (0.9 M Na⁺) at atemperature of between about 20° C. and about 35° C. (lower stringency),more preferably, between about 28° C. and about 40° C. (more stringent),and even more preferably, between about 35° C. and about 45° C. (evenmore stringent), with appropriate wash conditions. In particularembodiments, stringent hybridization conditions for DNA:RNA hybridsinclude hybridization at an ionic strength of 6×SSC (0.9 M Na⁺) at atemperature of between about 30° C. and about 45° C., more preferably,between about 38° C. and about 50° C., and even more preferably, betweenabout 45° C. and about 55° C., with similarly stringent wash conditions.These values are based on calculations of a melting temperature formolecules larger than about 100 nucleotides, 0% formamide and a G+Ccontent of about 40%. Alternatively, T_(m) can be calculated empiricallyas set forth in Sambrook et al., supra, pages 9.31 to 9.62. In general,the wash conditions should be as stringent as possible, and should beappropriate for the chosen hybridization conditions. For example,hybridization conditions can include a combination of salt andtemperature conditions that are approximately 20-25° C. below thecalculated T_(m) of a particular hybrid, and wash conditions typicallyinclude a combination of salt and temperature conditions that areapproximately 12-20° C. below the calculated T_(m) of the particularhybrid. One example of hybridization conditions suitable for use withDNA:DNA hybrids includes a 2-24 hour hybridization in 6×SSC (50%formamide) at about 42° C., followed by washing steps that include oneor more washes at room temperature in about 2×SSC, followed byadditional washes at higher temperatures and lower ionic strength (e.g.,at least one wash as about 37° C. in about 0.1×-0.5×SSC, followed by atleast one wash at about 68° C. in about 0.1×-0.5×SSC).

In one embodiment of the present invention, any of the amino acidsequences described herein, including homologues of such sequences(e.g., cysteine muteins), can be produced with from at least one, and upto about 20, additional heterologous amino acids flanking each of the C-and/or N-terminal end of the given amino acid sequence. The resultingprotein or polypeptide can be referred to as “consisting essentially of”a given amino acid sequence. According to the present invention, theheterologous amino acids are a sequence of amino acids that are notnaturally found (i.e., not found in nature, in vivo) flanking the givenamino acid sequence or which would not be encoded by the nucleotidesthat flank the naturally occurring nucleic acid sequence encoding thegiven amino acid sequence as it occurs in the gene, if such nucleotidesin the naturally occurring sequence were translated using standard codonusage for the organism from which the given amino acid sequence isderived. Similarly, the phrase “consisting essentially of”, when usedwith reference to a nucleic acid sequence herein, refers to a nucleicacid sequence encoding a given amino acid sequence that can be flankedby from at least one, and up to as many as about 60, additionalheterologous nucleotides at each of the 5′ and/or the 3′ end of thenucleic acid sequence encoding the given amino acid sequence. Theheterologous nucleotides are not naturally found (i.e., not found innature, in vivo) flanking the nucleic acid sequence encoding the givenamino acid sequence as it occurs in the natural gene.

The following examples are provided for the purpose of illustration andare not intended to limit the scope of the present invention.

Examples Example 1 Cloning and Expression of Human IFN-γ and CysteineMuteins of IFN-γ

A cDNA encoding human IFN-γ was amplified by RT-PCR from total RNAisolated from the human Jurkat T cell line (American Type CultureCollection, Rockville, Md.). The cells were grown in RPMI mediasupplemented with 10% FBS, 50 units/ml penicillin and 50 μg/mlstreptomycin. The cells were activated in vitro for 6 hours with 1 μg/mlPHA-L and 50 ng/ml PMA (phorbol 12-myristate 13-acetate), to induceIFN-γ expression prior to RNA isolation (Weiss et al., 1984; Wiskocil etal., 1985). RNA was isolated from the cells using an RNeasy Mini RNAisolation kit purchased from Qiagen, Inc. First strand synthesis ofsingle-stranded cDNA was accomplished using a 1st Strand cDNA SynthesisKit for RT-PCR (AMV) from Boehringer Mannheim Corp. (Indianapolis, Ind.)and random hexamers were used as the primer. Subsequent PCR reactionsusing the products of the first strand synthesis as template werecarried out with forward primer BB112 (5′-CGCAA GCTTG CCACC ATGAA ATATACAAGT TATAT C-3′; SEQ ID NO:3) and reverse primer BB113 (5′-CGCGG ATCCTCCGGA CTGGG ATGCT CTTCG ACCTT G-3′; SEQ ID NO:4). Primer BB112 annealsto the 5′ end of the coding sequence for the IFN-γ secretion signal andthe reverse primer, BB113, anneals to the 3′ end of the IFN-γ codingsequence. The resulting PCR product was digested with Hind III and BamHI, gel purified and cloned into pcDNA3.1(+) vector that had beendigested with Hind III and Bam HI, alkaline phosphatase treated, and gelpurified. A clone with the correct DNA sequence (Gray et al., 1982) wasdesignated pBBT192.

The gene for IFN-γ in pBBT192 was next modified by PCR for expression inE. coli. Primers were synthesized that anneal to the 5′ and 3′ ends ofthe gene, and incorporate sequences for translational initiation andtermination. The modification of the gene was performed in two steps.Forward primer BB1049 (5′CAG GAC CCA TAC GTA AAA GAA GCA GAA AAC CTTAAG; SEQ ID NO:5), and reverse primer BB1050 (5′CCG GAATTC TTA CTG GGATGC ACG TCG ACC TTG AAA CAG; SEQ ID NO:6) were used to incorporate a TAAtermination codon and EcoRI recognition site (GAATTC) at the 3′ end ofthe gene. The product of this PCR was then amplified using forwardprimer BB1048 (5′CGC GGA TCC ATC TTG GAG GAT GAT TAA ATG CAG GAC CCA TACGTA AAA G; SEQ ID NO:7) and reverse primer BB1050 to incorporate an ATGinitiation codon and a BamHI recognition site (GGATCC) for cloning intopET21a+, a T7 promoter expression vector (Novagen). The product of thissecond PCR was gel purified, digested with BamHI and EcoRI, repurified,and cloned into pUC19 that had been digested with BamHI and EcoRI,alkaline phosphatase (AP) treated, and purified using a Qiagen PCRpurification kit. A plasmid with the expected sequence was namedpBBT939. For expression experiments, this plasmid was introduced into E.coli strain BL21 (DE3) (Invitrogen).

Mutagenesis of IFN-γ was performed by PCR using methods described byScharf (1999) or by Higuchi (1999). For targeted residues whose codonswere close to the unique BsaI restriction site in pBBT939 at residuesE71 and T72, the inventors employed the method of Scharf, in which onemutagenic oligonucleotide and one PCR reaction was required. For othertargeted residues, Higuchi's method, using two mutagenicoligonucleotides, was employed. Higuchi's method requires three PCRs;two reactions that produce a “left” fragment and a “right” fragment, anda final “splicing” reaction, in which the left and right fragments aremixed and amplified with the two outside primers containing therestriction sites.

Similar methods were used to construct IFN gamma muteins N25C, K37C,Q67C and N83C, as well as an IFN gamma mutein, in which Q1 is deleted.Similar methods can be used to construct IFN gamma cysteine muteins Q1C,D2C, and P3C. Similar methods also can be used to construct cysteinemuteins in which a cysteine residue is inserted preceding the firstamino acid of the mature protein, Q1, or following the last amino acidof the protein, Q143. Cysteine analogs of IFN gamma are contemplated inthe context of the wild type gamma interferon sequence or in the contextof the sequences of other gamma interferon analogs, such as thosecontaining other amino acid substitutions, deletions or additions. Onepreferred set of gamma interferon analogs are those in which Q1, D2, P3,or a combination of these amino acids, are deleted.

PCR products were digested with either Eco RI and Bam HI, BamHI andBsaI, or EcoRI and BsaI, and cloned into similarly digested andAP-treated pBBT939. The newly cloned fragment was sequenced to confirmthat the desired cysteine substitution mutation was in place and that nounexpected mutations were present.

BamHI/EcoRI fragments encoding wild-type IFN-γ or IFN-γ cysteine muteinswere isolated after sequencing and ligated into BamHI/EcoRI/AP-digestedand purified pET21a+ (Novagen). The ligation reaction was transformedinto the E. coli strain JM109 (Promega). Plasmids fromampicillin-resistant colonies were analyzed for the correct size ofinsert, and transformed into BL21 (DE3) (Invitrogen). Expression ofnative IFN-γ and IFN-γ cysteine muteins results in the presence of anN-terminal methionine upstream of glutamine-1 (Q1) in the sequence.

Example 2 Eukaryotic Expression of Recombinant IFN-γ and the IFN-γCysteine Variants

IFN-γ and the IFN-γ cysteine muteins can also be expressed asintracellular or secreted proteins in eukaryotic cells such as yeast,insect cells or mammalian cells. Vectors for expressing the proteins andmethods for performing such experiments are described in catalogues fromvarious commercial supply companies such as Invitrogen, Inc.,Stratagene, Inc. and CloneTech, Inc. Procedures for expression proteinsin insect and mammalian cells are described in Cox et al., 2001.

Example 3 Small Scale Preparation of Wild Type IFN-γ and the IFN-γCysteine Variants in E. coli

The strains containing wild type IFN-γ or IFN-γ variants were grownovernight in Luria Broth (LB media) containing 100 μg/ml ampicillin and1% glucose at 37° C. in roll tubes. Saturated overnight cultures werediluted to an OD at 600 nm of −0.025 in the same media and incubated at37° C. in shake flasks. Typically a 400 ml culture was grown in a 2 Lshake flask. When culture ODs reached ˜0.5-0.6, IPTG was added to afinal concentration of 0.5 mM to induce expression of the IFN-γproteins. The cells were harvested by centrifugation, 3 hours postinduction and frozen at −20° C.

Cell pellets were thawed and treated with 10 ml of B-PER™ bacterialprotein extraction reagent according to the manufacturer's (Pierce)protocols. Other useful methods for lysing the cells includehomogenization and use of high pressure. The insoluble or aggregatedmaterial, which contains the bulk of the IFN-γ protein, was recovered bycentrifugation and resuspended in 10 ml B-PER™. This mixture was nexttreated with lysozyme (200 μg/ml) for 10 min to further disrupt the cellwalls, followed by MgCl₂ (10 mM final concentration) and protease-freeDNAse (2 μg/ml). Insoluble IFN-γ protein was collected by centrifugationand washed by resuspension in water and recentrifugation.

A variety of conditions for refolding the insoluble protein were testedin an experimental matrix format with wild type IFN-γ and the cysteinevariants. Solubilization reagents tested included chaotropic agents suchas urea and guanidine in the presence of a reducing agent(dithiothreitol, cysteine or TCEP [Tris(2-carboxyethyl)phosphine-HCl])and 20 mM Tris, base. For the renaturation step, the solubilizationswere diluted 20 fold with a buffered solution which contained one ormore of the following additives: glycerol (5-25%), arginine (100-500mM), Tween 20 (0.1-1%), guanidine (0.1-1 M), urea (0.1-3 M), coppersulfate (40 μmolar), and/or EDTA (1-2 mM). The refolds were left at 4°C. and analyzed by C4 Reversed Phase (RP) HPLC.

The greatest yield of refolded soluble IFN-γ resulted when the insolublematerial recovered from the detergent cell break was dissolved in 6 Mguanidine, 10 mM TCEP in 20 mM Tris, pH 8.0 where as wild type IFN-γcould be refolded in the absence of a reducing agent while the cysteinemuteins required the presence of a reducing agent. Other reducing agentssuch as cysteine, dithiothreitol, cysteamine, reduced glutathionine, orbeta-mercaptoethanol can be substituted for TCEP. This mixture wasstirred for 2 hours at room temperature, centrifuged to clarity, andgently diluted 28 fold into the renaturation solution consisting of 0.3M guanidine, 1 mM TCEP, 1 mM EDTA, and 20 mM Tris, pH 8.0. This refoldmixture was allowed to sit at 4° C. overnight without agitation.

Each refold (560 ml) was clarified by centrifugation and diluted 2× with20 mM Tris, 1 mM TCEP, pH 8.0 (S-Sepharose Buffer A). The mixture wasnext loaded onto a 10 ml S-Sepharose column (GE Biosciences)equilibrated in Buffer A. The column was eluted with a linear saltgradient (20 column volumes) from 25%-75% Buffer B (Buffer A+1 M NaCl).TCEP was used as the reducing agent in the column buffers because itdoes not interfere with the PEGylation reaction but does keep theprotein from forming disulfide bonds. Alternatively, other reducingagents (cysteine, dithiothreitol, cysteamine, reduced glutathione, betamercaptoethanol and related compounds) can be included in the columnbuffers and then subsequently removed by dialysis or chromatographybefore adding the PEG reagent. If a reducing agent such as cysteine (orcysteamine, dithiothreitol, reduced glutathione, beta mercaptoethanol ora dithiol (e.g., cystine, oxidized glutathione, cystamine) that can forma mixed disulfide when reduced) that is capable of forming a mixeddisulfide with the free cysteines in IFN— is used in the column buffersor refold and renaturation solutions, an additional reduction step isrequired before PEGylation.

The S-Sepharose fractions were assayed by SDS-PAGE. It should be notedthat when analyzed by SDS-PAGE, IFN-γ runs as a monomer due to thedisruption of the hydrophobic interactions that hold the IFN-γ dimertogether. Any disulfide-bonded material is reduced due to the presenceof TCEP in the S-Sepharose buffers. Fractions with substantially pureIFN-γ were pooled and frozen. Protein concentrations were measured usinga Bradford protein assay. Between 20-30 mgs of purified IFN-γ protein(depending on the construct) were typically recovered from a 400 mlshake flask culture.

Example 4 HPLC Assays for Characterization of the Wild Type and IFN-γCysteine Variants

A Reversed Phase (C4) HPLC method was developed for the purpose ofevaluating refold yields and the final purity of the recombinant IFN-γand the IFN-γ variants. A 50 μl aliquot of sample is applied to a C4Vydac (or similar C4 HPLC column) previously equilibrated in 60% BufferA (water+0.1% trifluoroacetic acid (TFA): 40% Buffer B(acetonitrile+0.1% TFA). The column is eluted with a 15 minute lineargradient from 40% Buffer B to 45% Buffer B. Absorbance is monitored at215 nm. A Beckman System Gold HPLC can be used for these experiments.IFN-γ elutes at approximately 6 minutes.

The purified IFN-γ proteins were also analyzed by Size Exclusion (SEC)HPLC to confirm that the proteins were dimeric. Samples of the proteinswere applied to a Bio-Sil SEC 400 (BioRad) HPLC column and eluted withan isocratic gradient of 1.2 M KCl, pH 7.5. BioRad MW standards wereused to calibrate the SEC HPLC column. The molecular weights for IFN-γmuteins and wild type IFN-γ were approximately 40 kDa as expected forthe homodimer.

Example 5 Amine PEGylation of IFN-γ

Wild type IFN-γ can also be PEGylated using amine reactive PEG reagents.Because the water hydroxyl anion of the aqueous buffer competes with theprimary amines, an excess of active PEG is usually needed, on the orderof 2 fold to 100 fold depending on the protein's reactivity. Thepredominant site(s) of PEGylation can be controlled based on the pH ofthe buffer. Generally, at pH values above 8.0, the epsilon-NH3 groupsreact first whereas at approximated pH 5-7, alpha-NH2 is the mostreactive.

For N-terminal PEGylation, IFN-γ is diluted into a buffer that hassufficient capacity to maintain the pH of the reaction between 5-7.Buffers containing primary amines such as Tris should be avoided. Theprotein's concentration can be on the order of 0.01 mg-50 mg/mL. PEG isadded on the order of 2- to 100-fold excess, preferably 2- to 10-foldexcess. The reaction is allow to sit overnight at 4° C. or until thereaction is considered complete. The PEGylated protein is separated fromthe non-PEGylated protein and the PEG reagents by column chromatographyusing ion exchange, hydrophobic interaction, or size exclusionchromatography. Chromatography can also be used to separate thePEGylated isoforms of the proteins that vary by the location and/or thenumber of PEGs attached. The proteins can be visualized by UV absorbanceat 280 nm whereas the PEG molecules can be identified by iodine assay(Sims et al., 1980).

For non-specific amine PEGylation the above reaction is run at a pHgreater than 8. The number of attachments desired in the final productcan be controlled by the amount of excess PEG reagent added and the timethat the reaction is allowed to proceed.

Example 6 Thiol Specific PEGylation of IFN-γ Cysteine Muteins

PEGylated IFN-γ can be prepared using a vinylsulfone or maleimide PEGreagents. Thiol specific PEG reagents ranging from 2-60 kDa and linearor branched are available from Nektar (USA), Nippon Oil and Fat (Japan)and Sunbio (South Korea).

Each IFN-γ cysteine mutein (0.5 mg) was diluted in 100 mM Tris, pH 8 toa final concentration of 100 μg/ml. Four aliquots of a 2.5× molar excessof 10 kDa maleimide-PEG were added at 30 min intervals (0, 30 min, 1 hrand 1.5 hr). After two hours at room temperature, the mixture wasdiluted with an equal volume of the 20 mM Tris, pH 8.0 and loaded onto a1 ml S-Sepharose column equilibrated in 20 mM Tris, pH 8.0 The PEGylatedprotein was eluted using a 20-60% gradient of buffer B (20 mM Tris, 1 MNaCl, pH 8.0) over 30 column volumes. The fractions were analyzed bySDS-PAGE. Fractions containing purified diPEG-Cys-IFN-γ (one PEG permonomer) were pooled and analyzed by the Bradford assay to determine theprotein concentration. DiPEG-Cys-IFN-γ also is referred to asPEG₂-Cys-IFN-γ.

The purified diPEGylated IFN-γ proteins were also analyzed by SEC-HPLCto confirm that the proteins had two PEGs attached and remained dimeric.PEGylation increases a protein's effective molecular weight more thanwould be expected based on the molecular weight of the PEG moiety due tothe water of hydration associated with the PEG group. As mentionedpreviously, a BioRad Bio-Sil 400 was used for SEC analysis. The columnwas calibrated using BioRad Gel permeation molecular weight markers.Based on SEC, the purified PEGylated IFN-γ proteins had apparentmolecular weights of ˜315 kDa as expected for IFN-γ dimer with two 10kDa PEGs attached. Similar experiments can be used to modify theproteins with different size PEGs (e.g. 20 kDa, 30 kDa, and 40 kDa PEGs,linear or branched).

Example 7 Method for Measuring the Bioactivities of Purified Wild TypeIFN-γ and the IFN-γ Cysteine Variants

The human ovarian cancer NIH OVCAR-3 cell line (ATCC) is sensitive tothe growth inhibiting properties of IFNs and can be used to measure thebioactivities of IFN-α, IFN-β and IFN-γ (Horoszewicz et al., 1979;Evinger and Pestka, 1981; our unpublished results). This assay measuresthe uptake and bioreduction of the tetrazolium salt MTS[3-(4,5-dimethylthiazol-2-yl)-5-3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium]and is proportional to cell number. In the presence of an electroncoupler such as phenazine methosulfate, MTS is converted to a formazanproduct that is soluble in tissue culture media and can be measureddirectly at 490 nm (Mosmann 1983). OVCAR cells are adherent cells andmaintained in MEM supplemented with 10% FBS, 50 units/ml Pen/Strep, and2 mM L-glutamine. Cells are passaged every 4 days in 1:2 dilutions.

For the bioassay, fifty μl (5×10³ cells) of the cell suspension werealiquoted per test well of a flat bottom 96 well tissue culture plate.Control wells contained media but no cells. Serial dilutions for theprotein samples and standard (IFN-γ, Endogen) were prepared in assaymedia. Fifty of the diluted protein samples were added to the test wellsin triplicate and the plates were incubated at 37° C. in a humidified 5%CO₂ tissue culture incubator for ˜7 days. On the 7th day, the media isremoved and the wells washed with PBS. 100 μl of phenol red free mediais added back to the wells along with 20 μl of an MTS/PMS mixture(CellTiter 96 Aqueous One Solution, Promega). Alternatively, the MTS/PMSsolution can be added directly to the test wells without washing. Theplates are incubated again at 37° C. in the tissue culture incubator for1-4 h. The plates are read at an absorbance of 490 nm using a microplatereader. Mean absorbance values for the triplicate control wells weresubtracted from mean values obtained for the test wells. An IC₅₀, theconcentration at half maximal inhibition, was calculated for each sampleto compare bioactivities of the proteins. The IFN-γ cysteine variants,and PEGylated forms of IFN-γ were assayed as described above. Ave IC₅₀values are shown in Table 2.

TABLE 2 Bioactivity and Yield Data for IFN-γ cysteine variants andPEGylated IFN-γ cysteine variants Region of Mean IC50s ± Activity MeanIC50s of Protein with SD relative PEG protein Activity IFN-γ Cysteine(ng/ml) to Wild PEGylation (ng/ml) relative to Construct Mutation (n ≧2) Type Efficiency (%) (n ≧ 2) Wild Type Wild type¹ — 81 ± 53 1.0 — — —Wild type² — 35 ± 7  2.2 — — — E38C B-C loop 90 ± 14 0.9 67 70 ± 42 1.1E39C B-C loop  30 2.7 47 130 ± 99  0.6 S40C B-C loop 250 ± 212 0.3 55425 ± 189 0.2 D63C C-D loop 200 0.4 50 85 ± 21 0.9 Q64C C-D loop 55 ± 211.5 95 40 ± 14 2.0 S65C C-D loop 90 ± 14 0.9 95 30 2.7 I66C C-D loop 250± 71  0.3 <20  400 ± 14  0.2 S84C D-E loop 75 ± 21 1.1 68 75 ± 35 1.1N85C D-E loop 250 ± 71  0.3 66 90 ± 14 0.9 K86C D-E loop 300 0.3 73 85 ±21 1.0 N97C Helix E  80 1.0 40 95 ± 7  0.9 V99C E-F loop 250 ± 71  0.320 400 ± 141 0.2 V100C E-F loop 350 ± 71  0.2 30 400  0.2 T101C E-F loop55 ± 7  1.4 50 25 ± 7  3.2 D102C E-F loop 200 0.4 50 85 ± 21 1.0 L103CE-F loop 45 ± 21 1.8 50  9 9 ¹Purchased from Endogen (Pierce Biotech)²Prepared in-house

In vitro bioactivities of certain IFN gamma muteins (N25C, K37C, Q67C,N83C and a mutein in which Q1 is deleted) were measured using the humanovarian cancer CAOV-3 cell line, which is available from the AmericanType Culture collection (Manassas, Va.; cat # HTB-75). The cells weremaintained in DMEM media supplemented with 10% fetal bovine serum,penicillin, streptomycin and Glutamax. For the bioassay, fifty μl (5×10³cells) of the cell suspension were aliquoted per test well of a flatbottom 96 well tissue culture plate. Control wells contained media butno cells. Serial dilutions for the protein samples and a WT IFN-gammastandard were prepared in assay media (phenol red-free DMEM mediasupplemented with 10% fetal bovine serum, 50 units/mL penicillin, 50micrograms/mL streptomycin and 2 mM Glutamax. Fifty μl of the dilutedprotein samples were added to the test wells in triplicate and theplates were incubated at 37° C. in a humidified 5% CO₂ tissue cultureincubator for 4 days. On day 4, 20 μl of CellTiter 96 Aqueous OneSolution (Promega) was added to each well and the plates incubated at37° C. in the tissue culture incubator for 1-4 h. The plates are read atan absorbance of 490 nm using a microplate reader. Mean absorbancevalues for the triplicate control wells were subtracted from mean valuesobtained for the test wells. IC₅₀ values were calculated for eachprotein and are shown in Table 3. The data indicate that in vitrobioactivities of the muteins were comparable to that of wild type IFNgamma. IC₅₀s for the proteins were within 2-fold of the IC₅₀ for wildtype IFN gamma. Q1 is thought to be important for biological activity ofinterferon gamma; thus it was unexpected to find that a mutant lackingQ1 had similar biological activity as wild type interferon gamma.

TABLE 3 IC₅₀s of IFN gamma muteins for inhibiting growth of human CAOV-3cells in vitro. Region of Protein with IC₅₀ IFN Gamma Mutein CysteineMutation (ng/mL) WT IFN gamma — 1.4 ± 0.7 Q1 deletion — 0.7 ± 0.2 N25CA-B loop 2.2 ± 0.5 K37C B-C loop 0.8 ± 0.2 Q67C C-D loop 2.3 ± 0.1 N83CD-E loop 1.5 ± 0.5

Example 8 Pharmacokinetic Experiments to Demonstrate IncreasedCirculating Half-Lives of PEG₂-Cys-IFN-γ Relative to unPEGylated IFN-γ

Rats (3/group) were given a single intravenous (IV) injection (100μg/kg) of wild type IFN-γ or 10 kDa PEG₂-Cys-IFN-γ (S84C). Blood samplesare drawn at various times and frozen. A commercially available IFN-γELISA kit (R&D systems) was used to measure serum levels of the IFN-γproteins. Alternatively, quantitative Westerns blots can be run todetermine serum levels. Terminal pharmacokinetic parameters werecalculated using WinNonlan software and non-compartmental analysis. Thecirculating half-lives were calculated to be 1.2 hr for wild type IFN-γand 9.5 hr for 10 kDa PEG₂-Cys-IFN-γ (S84C). These data indicate thatthe half-lives of the PEGylated proteins were much longer than thehalf-life of wild type IFN gamma.

In an analogous experiment, rats (3/group) were given single aintraperitoneal (ip) injection of wild type IFN-γ, 10 kDa PEG₂-Cys-IFN-γ(L103C), 20 kDa PEG₂-Cys-IFN-γ (L103C) or 40 kDa PEG₂-Cys-IFN-γ (L103C).Blood samples were drawn at various times and analyzed by the ELISAassay. The circulating half-lives were calculated to be <1 hr for wildtype IFN-γ, 10.3 hr for 10 kDa PEG₂-Cys-IFN-γ, 17.1 hr for 20 kDaPEG₂-Cys-IFN-γ and 21.7 hr for 40 kDa PEG₂-Cys-IFN-γ. These dataindicate that the half-lives of the PEGylated proteins were much longerthan the half-life of wild type IFN gamma.

Example 9 Evaluation of PEGylated IFN-γ's In Vivo Activity in XenograftTumor Model

The relative efficacies of the PEGylated IFN-γ variants as an anti-tumordrug can be evaluated in a human tumor xenograft growth using humantumor cell lines, such as NIH-OVCAR-3 cells. in athymic nude mice (Burkeet al., 1999, Malik et al., 1991). Inhibition of human tumor xenograftgrowth in nude mice occurs through a direct anti-proliferative effect onthe human tumor cells. Since these mice cannot mount an immune response,potential immunogenicity problems resulting from use of human IFN-γproteins in mice will not be an issue.

Athymic nude mice can be purchased from a commercial vendor such asCharles River Laboratories. Each mouse is injected intraperitoneally orsubcutaneously with 1-5×10⁶ NIH-OVCAR-3 tumor cells on day 0 andrandomly assigned to test groups, consisting of ten mice each. Sevendays later the different test groups receive ip, sc or iv injections ofhuman wild type IFN-γ, PEG₂-Cys-IFN-γ or placebo (vehicle solution) atspecified intervals: every day (ED), every other day (EOD) or everythird day (ETD) for 8-10 weeks. Doses of the proteins required for tumorgrowth inhibition can be determined experimentally and are expected tobe in the range of 5 μg/mouse/injection to 5 mg/mouse/injection. Tumorvolumes in the test groups are measured over time. At time of sacrifice,autopsies are performed to evaluate tumor burden and tumor weight. Weexpect that the tumors in the animals treated with PEG₂-Cys-IFN-γ willbe smaller than the placebo and wild type IFN-γ groups.

We compared the ability of IFN-gamma and the 40 kDa-PEG-L103C protein toinhibit growth of human NIH:OVCAR-3 tumor cells transplanted intoathymic nude mice. Female athymic nude (nu/nu) mice were obtained fromCharles River (Wilmington, Mass.). The NIH:OVCAR-3 cells were grown invitro, harvested by trypsinization, washed and resuspended at aconcentration of 25×10⁶ cells per mL in a 50:50 mixture of MEMmedia/Matrigel. Matrigel was purchased from BD Biosciences (Bedford,Mass.). Animals were randomized to different test groups according tobody weight on Day 0. Mice (10/group) received subcutaneous injectionsof 0.2 mL of the MEM media/Matrigel mixture containing 5×10⁶ cells inthe axillary area on Day 0. Animals received subcutaneous injections(0.2 mL/animal) of the test compounds beginning on Day 1 and continuingthrough Day 70 using a 3×/week dosing schedule (Monday, Wednesday,Friday). Mice were weighed on Day 0, prior to the injection of cells,and on the days of tumor measurements. Tumors were measured weekly usingcalipers for 10 weeks post injection. Tumor volume was determined usinga formula of ((width×width)×length)/2). The different treatment groupsreceived injections of vehicle solution [phosphate buffered salinecontaining 0.1 mg/mL of mouse albumin (Sigma-Aldrich, Inc., St. Louis,Mo.)], wild type IFN-gamma in vehicle solution (15 μg/injection/mouse)or 40 kDa-PEG-L103C in vehicle solution (15 μg/injection/mouse). Wildtype IFN-gamma was prepared by us.

Final tumor volumes and weights at necropsy (on day 70) are summarizedin Table 4. Mean tumor volumes of animals treated with wild typeIFN-gamma were, on average, larger than tumors in animals treated withvehicle solution, indicating that there is no benefit of wild typeIFN-gamma versus placebo for inhibiting tumor growth at this dose anddosing frequency. Final mean tumor volumes in animals treated with wildtype IFN-gamma and vehicle solution were not statistically differentfrom one another. In contrast, mean tumor volumes in animals treatedwith PEG-L103C were 51% smaller than mean tumor volumes in animalstreated with vehicle solution and 68% smaller than mean tumor volumes inanimals treated with wild type IFN-gamma. The differences between meantumor volumes in animals receiving PEG-L103C and vehicle or wild typeIFN-gamma were statistically significant (p<0.01). Mean tumor weights atnecropsy (Day 70) were significantly reduced in animals receivingPEG-L03C compared to mean tumor weights in animals receiving injectionsof vehicle solution (53% reduction) or wild type IFN-gamma (69%reduction).

TABLE 4 Tumor volumes and weights in mice treated with Vehicle, wildtype IFN-gamma and PEG-L103C at study termination on day 70. Data aremeans ± SD for 10 mice per group. Tumor Volume Tumor Weight Compound(mm³) (g) 1. Vehicle   949 ± 139 0.939 ± 0.136 2. WT IFN-gamma 1,475 ±280 1.424 ± 0.280 3. PEG-L103C   466 ± 89 *    0.444 ± 0.063 * * p <0.01 vs Vehicle and wild type IFN-gamma

Example 10 Evaluation of PEGylated IFN-γ's In Vivo Activity atStimulating the Immune System In Vivo

The efficacy of PEG₂-Cys-IFN-γ as an anti-infective drug, can also betested in a mouse model of chronic granulomatous disease (CGD). A mouseknockout model of CGD has been developed by Jackson et al. (1995) thathas a targeted disruption of the p47phox gene. Identical to human CGD,leukocytes from p47phox−/− mice produce no superoxide and are unable tokill staphylococci efficiently. The p47phox−/− mice develop lethalinfections and granulomatous inflammation similar to those encounteredin human CGD patients. Researchers have investigated the prophylacticeffect of IFN-γ in the CGD mouse model p47phox form of CGD. In an animalstudy, murine IFN-γ, (20,000 U) or placebo was administeredsubcutaneously to p47phox−/− mice. By 6 weeks of study, there were 3infections in the IFN-γ treated group (n=60) as compared with 13infections in the placebo group (n=58) (77% reduction in infections,p<0.01).

For this study, it is necessary to prepare the murine version of aPEG-IFN-γ cysteine mutein since there is no cross-reactivity betweenspecies. Murine IFN-γ is commonly used in mouse models of infectiousdiseases (Jackson et al., 2001; Murray, 1990; Flynn et al., 1993).

Like its human counterpart, murine IFN-γ exists as a glycosylatednoncovalent homodimer with similar secondary and tertiary structures.Therefore, it is relatively straightforward to transfer the thiolspecific PEGylation technology from the human protein to the mouseprotein. FIG. 3 of Ealick et al. (1993) shows the alignment of the humanand mouse gamma interferon amino acid sequences, which can be used tochoose appropriate mouse amino acids to change to cysteine residues.Murine-human IFN-γ hybrids have been made to identify the regions thatare responsible for species specificity recognition of human IFN-γ withthe human IFN-γ receptor (Lundell et al., 1994). In particular, itappears that amino acids in the A-B loop (more specifically residues20-23) and His 111 in Helix F play critical roles in species specificreceptor interactions. Several papers describe the expression, refoldingand purification of murine IFN-γ (Nagata et al., 1886; Zavodny et al.,1988; Lundell et al., 1994). The amino acid sequence of murine IFN-γ isgiven in Sequence ID No. 2. When expressed in E. Coli, the protein maycontain an N-terminal methionine not found in the native protein. MouseIFN gamma contains a C-terminal cysteine residue. This cysteine residuecan be deleted or substituted with another amino acid, preferably,alanine, serine or threonine, to facilitate expressions and purificationof the protein.

The specific PEG₂-Cys-IFN-γ to be used for these experiments (10, 20, or40 kDa PEG) can be determined by the results of the rat PK experimentsdescribed above and their relative activities in the in vitro bioassay.The knockout mice are commercially available from Taconic (Albany, N.Y.)in collaboration with the National Institute of Allergy and Infectiousdiseases (NIAD) exchange program. Before the study begins the animalsare maintained on a broad-spectrum antibiotic. At the initiation of theexperiment, the antibiotics are removed from their water, making themsusceptible to infection. The animals are monitored three times weeklyfor gross evidence of infection for three months.

Example 11 Evaluation of PEGylated IFN-γ's In Vivo Activity atInhibiting Viral Infections

The ability of PEG₂-Cys-IFN-γ proteins to inhibit viral replication canbe determined using in vitro viral assays. The bioactivities of thecysteine muteins and the PEG₂-Cys-IFN-γ proteins can be measured usingan in vitro viral cytopathic effect inhibition assay (Arakawa et al.,1985). Human HEp2 or WISH cells (ATCC) are plated in 96-well plates(3×10⁴ cells/well) and grown to near confluency at 37° C. The cells arewashed and treated for 24 hr with serial dilutions of the test proteins.Controls include no IFN-γ, wild type IFN-γ prepared by us and wild typeIFN-γ purchased from a commercial vendor. Encephalomyocarditis virus(EMCV) is added at a multiplicity of infection of 0.1 and the platesincubated for a further 24-48 hr at 37° C. Additional controls includesamples without virus. When 90% or more of the cells have been killed inthe virus-treated, no IFN-γ control wells (determined by visualinspection of the wells), an MTS solution is added to the wells.Absorbance of the wells is read at 490 nm after 1-4 hr. Samples arenormally analyzed in triplicate. IC₅₀ values can be used to compare therelative potencies of the PEG₂-Cys-IFN-γ cysteine muteins. The IC₅₀value for commercially available IFN-γ in this assay is typicallybetween 0.05-0.15 ng/ml.

Example 12 Glucagon

Glucagon is a small peptide that is secreted by intestinal cells andacts primarily on the liver to stimulate glucose production andsecretion. Glucagon is important for counteracting hypoglycemia. Theamino acid sequence of glucagon is represented herein by SEQ ID NO:8. Acysteine residue can be added preceding the first amino acid, H1, orfollowing the last amino acid T29. A cysteine residue also can beintroduced into the protein by substitution for at least one amino acidselected from the group consisting of H1, S2, Q3, G4, T5, F6, T7, S8,D9, Y10, S11, K12, Y13, L14, D15, S16, R17, R18, A19, Q20, D21, F22,V23, Q24, W25, L26, M27, N28, T29. These amino acid changes can be madein the context of native sequence glucagons or in the context ofglucagon analogs, such as those described in U.S. Pat. No. 5,510,459.The cysteine variants of the present invention can be modified withcysteine-reactive moieties such as cysteine reactive PEGs. Thecysteine-modified peptides can be purified from unmodified peptides byany of a number of methods known in the art such as columnchromatography.

Alternatively, glucagon can be modified with PEG at K12 using anamine-reactive PEG reagent. Alternatively, K12, can be changed to anon-lysine residue, preferably arginine, and a new lysine residueintroduced into the protein. An amine-reactive PEG can then be attachedto the added lysine residue. The added lysine residue can be insertedpreceding the first amino acid, H1, or following the last amino acidT29. A lysine residue also can be added to the protein by substitutionfor at least one amino acid selected from the group consisting of H1,S2, Q3, G4, T5, F6, T7, S8, D9, Y10, S11, Y13, L14, D15, S16, R17, R18,A19, Q20, D21, F22, V23, Q24, W25, L26, M27, N28, T29. These amino acidscan be made in the context of native sequence glucagons or in thecontext of glucagon analogs, such as those described in U.S. Pat. No.5,510,459. The lysine variants of the present invention can be modifiedwith amine-reactive moieties such as amine-reactive PEGs. Theamine-modified peptides can be purified from unmodified peptides by anyof a number of methods known in the art such as column chromatography.

Glucagon also can be modified at its N-terminus using amine-reactivemoieties using methods well known in the art.

The modified glucagon proteins can be tested for in vitro and in vivobiological activity using methods known in the art.

Example 12 GLP-1

Glucagon-like peptide-1 (GLP-1) is a 31 amino acid peptide synthesizedprimarily by the L-cells of the intestine in response to nutrientingestion. The protein also exists as a 30 amino acid form, called theamide form, in which G31 is absent. The protein is expressed as part ofa larger polypeptide, proglucagon, that is proteolytically cleaved toyield GLP-1, glucagon, oxyntomodulin (OXM) and glucagon-like peptide-2(GLP-2), and several additional peptides. The amino acid sequence ofGLP-1 is conserved among mammals. GLP-1 has several properties thatsuggest it may prove useful for treating diabetes and obesity. GLP-1acts on the pancreas to stimulate proinsulin gene expression, insulinsecretion and inhibit glucagon secretion. These effects of GLP-1 areglucose-dependent; thus GLP-1 may provide a safe mechanism for loweringelevated blood glucose levels such as is seen in diabetes. GLP-1 alsostimulates growth and proliferation of pancreatic beta cells, which arethe pancreatic cells responsible for synthesizing insulin. Thus, GLP-1could be used to increase beta cell mass or increase insulin secretionpotential in patients with diabetes. GLP-1 also reduces caloric intake,reduces hunger sensations, and slows gastric emptying and acidsecretion, suggesting that it may prove useful as an anti-obesity drug.Since many type 2 diabetics are obese, GLP-1 could provide a dualmechanism for improving the physiology of these patients, both bydecreasing weight and improving their ability to synthesize and utilizeinsulin.

The amino acid sequence of GLP-1 is represented herein by SEQ ID NO:9. Acysteine residue can be added preceding the first amino acid, H1, orfollowing the last amino acid G31. A cysteine residue also can beintroduced into the protein by substitution for at least one amino acidselected from the group consisting of H1, A2, E3, G4, T5, F6, T7, S8,D9, V10, S11, S12, Y13, L14, E15, G16, Q17, A18, A19, K20, E21, F22,I23, A24, W25, L26, V27, K28, G29, R30 and G31. These amino acid changescan be made in the context of the 1-30 amino acid form of GLP-1 or the1-31 amino acid form of GLP-1. These amino acids can be made in thecontext of GLP-1 analogs, such as those described in U.S. Pat. No.6,458,924, U.S. Pat. No. 6,620,910, U.S. Pat. No. 6,703,365, U.S. Pat.No. 7,041,646, U.S. Pat. No. 7,067,488, U.S. Pat. No. 7,101,843, U.S.Pat. No. 7,138,486, U.S. Pat. No. 7,199,217, and U.S. Pat. No.7,211,557. A preferred GLP-1 analog is one that inhibits cleavage of H1and A2 from the peptide by proteases. A preferred GLP-1 analog is one inwhich A2 is changed to a non-alanine amino acid, preferably serine,glycine or cysteine. Another preferred GLP-1 analog is one in which E3is substituted with an a non-glutamic acid amino acid such as proline,which prevents proteolytic cleavage of H1 and A2 from the peptide. Thecysteine variants of the present invention can be modified withcysteine-reactive moieties such as cysteine reactive PEGs. Thecysteine-modified peptides can be purified from unmodified peptides byany of a number of methods known in the art such as columnchromatography.

Alternatively, GLP-1 can be modified with PEG at one or both of thelysine residues K20 and K28. To make the PEG reaction specific for aparticular lysine residue, K20 or K28 can be changed to a non-lysineamino acid, preferably arginine, and the mutant protein modified withPEG at the remaining lysine residue. Additionally, both lysine residues,K20 and K28, can be changed to non-lysine residues and a new lysineresidue introduced into the protein. The new lysine residue can be addedpreceding the first amino acid, H1, or following the last amino acidG31. A lysine residue also can be introduced into the protein bysubstitution for at least one amino acid selected from the groupconsisting of H1, A2, E3, G4, T5, F6, T7, S8, D9, V10, S11, S12, Y13,L14, E15, G16, Q17, A18, A19, E21, F22, I23, A24, W25, L26, V27, G29,R30 and G31. These amino acid changes can be made in the context of the1-30 amino acid form of GLP-1 or the 1-31 amino acid form of GLP-1.These amino acids can be made in the context of GLP-1 analogs, such asthose described in the preceding paragraph. The lysine variants of thepresent invention can be modified with amine-reactive moieties such asamine-reactive PEGs. The amine-modified peptides can be purified fromunmodified peptides by any of a number of methods known in the art suchas column chromatography.

GLP-1 also can be modified at its N-terminus using methods described inthe art. The modified GLP-1 proteins can be tested for in vitro and invivo biological activity using methods well known in the art.

Example 13 GLP-2

GLP-2 is a small peptide secreted by intestinal cells. GLP-2 actslocally within the intestine to stimulate intestinal crypt cellproliferation, inhibit intestinal cell apoptosis and expand the mucosalepithelium. GLP-2 also promotes nutrient absorption. The amino acidsequence of GLP-2 is represented herein by SEQ ID NO:10. A cysteineresidue can be added preceding the first amino acid, H1, or followingthe last amino acid D33. A cysteine residue also can be introduced intothe protein by substitution for at least one amino acid selected fromthe group consisting of H1, A2, D3, G4, S5, F6, S7, D8, E9, M10, N11,T12, I13, L14, D15, N16, L17, A18, A19, R20, D21, F22, I23, N24, W25,L26, I27, Q28, T29, K30, I31, T32, and D33. These amino acids can bemade in the context of native sequence GLP-2 or in the context of GLP-2analogs, such as those described in U.S. Pat. No. 7,112,567, U.S. Pat.No. 7,056,886, and U.S. Pat. No. 5,952,301. A preferred GLP-3 analog isone that inhibits cleavage of H1 and A2 from the peptide by proteases. Apreferred GLP-2 analog is one in which A2 is changed to a non-alanineamino acid, preferably serine, glycine or cysteine. Another preferredGLP-2 analog is one in which D3 is substituted with a non-aspartic acidamino acid such as proline, which prevents proteolytic cleavage of H1and A2 from the peptide. The cysteine variants of the present inventioncan be modified with cysteine-reactive moieties such as cysteinereactive PEGs. The cysteine-modified peptides can be purified fromunmodified peptides by any of a number of methods known in the art suchas column chromatography.

Alternatively, GLP-1 can be modified with an amine-reactive moiety atK30. Alternatively, K30, can be changed to a non-lysine residue,preferably arginine, and a new lysine residue introduced into theprotein. An amine-reactive moiety can then be attached to the newlyadded lysine residue. The new lysine residue can be inserted precedingthe first amino acid, H1 or following the last amino acid D33. A lysineresidue also can be added to the protein by substitution for at leastone amino acid selected from the group consisting of H1, A2, D3, G4, S5,F6, S7, D8, E9, M10, N11, T12, I13, L14, D15, N16, L17, A18, A19, R20,D21, F22, I23, N24, W25, L26, I27, Q28, T29, I31, T32, D33. These aminoacids can be made in the context of native sequence GLP-2 or GLP-2analogs, such as those described in U.S. Pat. No. 7,112,567, U.S. Pat.No. 7,056,886, and U.S. Pat. No. 5,952,301. A preferred GLP-2 analog isone in which A2 is changed to a non-alanine amino acid, preferablyserine or glycine. The lysine variants of the present invention can bemodified with amine-reactive moieties such as amine-reactive PEGs. Theamine-modified peptides can be purified from unmodified peptides by anyof a number of methods known in the art such as column chromatography.

GLP-2 also can be modified at its N-terminus using methods well known inthe art. The modified GLP-2 proteins can be tested for in vitro and invivo biological activity using methods well known in the art.

Example 14 Exendin-4

The amino acid sequence of exendin-4 is represented herein by SEQ IDNO:11. Exendin is a GLP-1 agonist and exerts similar functions as GLP-1.A cysteine residue can be inserted preceding the first amino acid, H1,or following the last amino acid S38. A cysteine residue also can beintroduced into the protein by substitution for at least one amino acidselected from the group consisting of H1, G2, E3, G4, T5, F6, T7, S8,D9, L10, S11, K12, W13, M14, E15, E16, E17, A18, V19, R20, L21, F22,I23, E24, W25, L26, K27, N28, G29, G30, P31, S32, S33, G34, A35, P36,P37, and S38. These amino acids can be made in the context of nativesequence exendin-4 of exendin-4 analogs, such as those described in U.S.Pat. No. 6,767,887, U.S. Pat. No. 6,924,264, U.S. Pat. No. 7,115,569,U.S. Pat. No. 7,153,825, and U.S. Pat. No. 7,157,555. The cysteinevariants of the present invention can be modified with cysteine-reactivemoieties such as cysteine reactive PEGs. The cysteine-modified peptidescan be purified from unmodified peptides by any of a number of methodsknown in the art such as column chromatography.

Alternatively, exendin-4 can be modified with an amine-reactive moietyat one or both of the lysine residues K12 and K27. To make the reactionspecific for a particular lysine residue, K12 or K27 can be changed to anon-lysine amino acid, preferably arginine, and the mutant proteinmodified with an amine-reactive moiety at the remaining lysine residue.Additionally, both lysine residues, K12 and K27, can be changed tonon-lysine residues, preferably arginine residues, and a new lysineresidue introduced elsewhere in the protein. The new lysine residue canbe added preceding the first amino acid, H1, or following the last aminoacid S38. A lysine residue also can be introduced into the protein bysubstitution for at least one amino acid selected from the groupconsisting of H1, G2, E3, G4, T5, F6, T7, S8, D9, L10, S11, W13, M14,E15, E16, E17, A18, V19, R20, L21, F22, I23, E24, W25, L26, N28, G29,G30, P31, S32, S33, G34, A35, P36, P37, and S38. These amino acidchanges can be made in the context of native sequence exendin-4 or inthe context of exendin-4 analogs, such as those described in U.S. Pat.No. 6,767,887, U.S. Pat. No. 6,924,264, U.S. Pat. No. 7,115,569, U.S.Pat. No. 7,153,825, and U.S. Pat. No. 7,157,555. The lysine variants ofthe present invention can be modified with amine-reactive moieties suchas amine-reactive PEGs. The amine-modified peptides can be purified fromunmodified peptides by any of a number of methods known in the art suchas column chromatography.

Exendin-4 also can be modified at its N-terminus using methods describedin the art. The modified exendin-4 proteins can be tested for in vitroand in vivo biological activity using methods known in the art.

Example 15 PYY

Peptide YY (PYY) is a small peptide secreted by the intestine. PYYfunctions to control food intake. The amino acid sequences of human PYYis represented herein by SEQ ID NO:12. A truncated form of PYY in whichamino acids 1 and 2 are deleted is referred to as PYY 3-36. Analternative form of PYY includes a P at position 34 (P34). A cysteineresidue can be added preceding the first amino acid, Y1, or followingthe last amino acid Y36. A cysteine residue can be added preceding thefirst amino acid, 13, of the PYY 3-36 form of the peptide. A cysteineresidue also can be introduced into the protein by substitution for atleast one amino acid selected from the group consisting of Y1, P2, I3,K4, P5, E6, A7, P8, G9, E10, D11, A12, S13, P14, E15, E16, L17, N18,R19, Y20, Y21, A22, S23, L24, R25, H26, Y27, L28, N29, L30, V31, T32,R33, Q34, R35, and Y36. These amino acids can be made in the context ofnative sequence PYY or PYY 3-36 or in the context of PYY or PYY 3-36analogs such as those described in U.S. Pat. No. 6,046,167, U.S. Pat.No. 5,328,899, U.S. Pat. No. 5,026,685, and U.S. Pat. No. 7,186,692. Thecysteine variants of the present invention can be modified withcysteine-reactive moieties such as cysteine reactive PEGs. Thecysteine-modified peptides can be purified from unmodified peptides byany of a number of methods known in the art such as columnchromatography.

PYY also can be modified with an amine-reactive moiety at the N-terminalY1 amino acid or at the K4 amino acid. Alternatively, K4 can be changedto a non-lysine amino acid, preferably arginine, and a new lysineresidue introduced into the protein. The new lysine residue can be addedpreceding the first amino acid, Y1, or following the last amino acidY31. A lysine residue also can be introduced into the protein bysubstitution for at least one amino acid selected from the groupconsisting of Y1, P2, I3, P5, E6, A7, P8, G9, E10, D11, A12, S13, P14,E15, E16, L17, N18, R19, Y20, Y21, A22, S23, L24, R25, H26, Y27, L28,N29, L30, V31, T32, R33, Q34, R35, and Y36. These amino acids can bemade in the context of native sequence PYY or PYY 3-36 or in the contextof PYY or PYY 3-36 analogs, such as those described in U.S. Pat. No.6,046,167, U.S. Pat. No. 5,328,899, U.S. Pat. No. 5,026,685, and U.S.Pat. No. 7,186,692. The lysine variants of the present invention can bemodified with amine-reactive moieties such as amine-reactive PEGs. Theamine-modified peptides can be purified from unmodified peptides by anyof a number of methods known in the art such as column chromatography.

PYY or PYY 3-36 also can be modified at its N-terminus using methodsdescribed in the art. The modified PYY and PYY 3-36 peptides can betested for in vitro and in vivo biological activity using methods wellknown in the art.

Example 16 Ghrelin

Ghrelin is a 28 amino acid peptide that was originally isolated from thestomach. The amino acid sequence of Ghrelin is represented herein by SEQID NO:13. Ghrelin circulates in the body in both an acylated form(AC-ghrelin) and a non-acylated (NA-ghrelin) form, with the non-acylatedform predominating under most situations. AC-ghrelin has an n-octanoicfatty acid attached to the hydroxyl group of Ser3. AC-ghrelin binds a Gprotein coupled receptor, GHSR1a, which is preferentially expressed inthe pituitary and hypothalamus and is distinct from the growth hormonereleasing factor receptor. The GHSR1a receptor also is expressed in avariety of tissues, including adrenal and thyroid gland, heart, liver,kidney and skeletal muscle. NA-ghrelin can bind a differentuncharacterized receptor that is expressed in heart muscle and certainother tissues. AC-ghrelin is a naturally occurring growth hormone (GH)secretagogue. Administration of the AC-ghrelin to animals and humansincreases circulating GH, prolactin, ACTH and cortisol levels, decreasesserum insulin levels, increases plasma glucose levels, in causeshyperglycemia. NA-ghrelin does not cause these changes. NA-ghrelinappears to inhibit the endocrine functions of ghrelin, but not theneuroendocrine functions of AC-ghrelin. NA-ghrelin inhibits theappetite-stimulating effects of AC-ghrelin in humans. NA-ghrelin alsoinhibits apoptosis of cardiac mycotyes. Both AC-ghrelin and NA-ghrelinshare certain functions such as cardiovascular actions, and may beuseful for treating heart disease. Both AC-ghrelin and NA-ghrelininhibit cell death in cardiomyocytes and endothelial cells, and bothpeptides stimulate proliferation of osteoblast cell lines in vitro.NA-ghrelin has the potential to function as an anti-obesity drug inpeople. NA-ghrelin also slows emptying of gastric contents. AC-ghrelinstimulates appetite in people and may prove useful for treatingmetabolic and eating disorders such as cachexia and anorexia. Ghrelinsecretion is stimulated by fasting and energy restriction, and inhibitedby food intake, glucose, insulin and somatostatin. Ghrlein levels areincreased in anorexia and cachexia and decreased in obesity.

In short term studies mice treated with NA-ghrelin decreased food intakeand slowed the rate of gastric emptying. These physiological changeswere mediated through NA-ghrelin action on the hypothalamus. Thus,NA-ghrelin appears to cause a negative energy balance in the body.Transgenic mice overexpressing NA-ghrelin displayed decreased lineargrowth, lower food intake, and decreased body weight. These micedisplayed lower serum IGF-I and GH levels compared to control mice.NA-ghrelin transgenic mice responded to GHRH but displayed a bluntedresponse to AC-ghrelin administration, suggesting that NA-ghrelin canfunction as a specific AC-ghrelin antagonist. NA-ghrelin transgenic micestill responded normally to GH releasing hormone, indicating that theantagonist effect was specific for AC-ghrelin and not all GH releasinghormones.

Ghrelin has a circulating half-life of 27-31 min following intravenousadministration to humans. AC-ghrelin has a shorter half-life of 9-13min, possibly due to more rapid degradation.

A cysteine residue can be inserted preceding the first amino acid, G1,or following the last amino acid R28 of ghrelin. A cysteine residue alsocan be introduced into the protein by substitution for at least oneamino acid selected from the group consisting of G1, S2, S3, F4, L5, S6,P7, E8, H9, Q10, R11, V12, Q13, Q14, R15, K16, E17, S18, K19, K20, P21,P22, A23, K24, L25, Q26, P27, and R28. These amino acids can be made inthe context of acylated or nonacylated ghrelin. These amino acid changescan be made in the context of ghrelin analogs, such as those describedin U.S. Pat. No. 7,176,292, U.S. Pat. No. 7,115,767, U.S. Pat. No.7,074,562 and U.S. Pat. No. 6,967,237. The cysteine variants of thepresent invention can be modified with cysteine-reactive moieties suchas cysteine reactive PEGs. The cysteine-modified peptides can bepurified from unmodified peptides by any of a number of methods known inthe art such as column chromatography.

Alternatively, Ghrelin can be modified with an amine-reactive moiety atthe N-terminus or at one or all of the lysine residues K16, K19, K20 andK24. To make the modification reaction specific for a particular lysineresidue, K16, K19, K20 or K24 can be changed to a non-lysine amino acid,preferably arginine, and the mutant protein modified with anamine-reactive moiety at the remaining lysine residue. Additionally, allof the lysine residues, K16, K19, K20 and K24, can be changed tonon-lysine residues, preferably arginine residues, and a new lysineresidue introduced into the protein. The new lysine residue can be addedpreceding the first amino acid, G1, or following the last amino acidR28. A lysine residue also can be introduced into the protein bysubstitution for at least one amino acid selected from the groupconsisting of G1, S2, S3, F4, L5, S6, P7, E8, H9, Q10, R11, V12, Q13,Q14, R15, E17, S18, P21, P22, A23, L25, Q26, P27, and R28. These aminoacids can be made in the context of other Ghrelin analogs, such as thosedescribed in U.S. Pat. No. 7,176,292, U.S. Pat. No. 7,115,767, U.S. Pat.No. 7,074,562 and U.S. Pat. No. 6,967,237. The lysine variants of thepresent invention can be modified with amine-reactive moieties such asamine-reactive PEGs. The amine-modified peptides can be purified fromunmodified peptides by any of a number of methods known in the art suchas column chromatography.

Ghrelin also can be modified at its N-terminus using methods describedin the art. The modified ghrelin proteins can be tested for in vitro andin vivo biological activity using methods well known in the art.

Example 17 Oxyntomodulin

Oxyntomodulin (OXM) is a small peptide secreted by the L cells of theintestine. OXM serves to decrease food intake and decrease weight gain,and thus may prove useful for treating obesity. The amino acid sequenceof OXM is represented herein by SEQ ID NO:14. OXM is a derivative ofglucagon in which the sequence DTKRNKNNIA (SEQ ID NO:15) is added to thefirst 27 amino acids of glucagon. A cysteine residue can be addedpreceding the first amino acid, H1, or following the last amino acidA38. A cysteine residue also can be introduced into the protein bysubstitution for at least one amino acid selected from the groupconsisting of H1, S2, Q3, G4, T5, F6, T7, S8, D9, Y10, S11, K12, Y13,L14, D15, S16, R17, R18, A19, Q20, D21, F22, V23, Q24, W25, L26, M27,N28, D29, T30, K31, R32, N33, K34, N35, N36, I37, A38. These amino acidscan be made in the context of native sequence OXM or OXM analogs such asthose described in U.S. Pat. No. 5,858,975. The cysteine variants of thepresent invention can be modified with cysteine-reactive moieties suchas cysteine reactive PEGs. The cysteine-modified peptides can bepurified from unmodified peptides by any of a number of methods known inthe art such as column chromatography.

Alternatively, OXM can be modified with an amine-reactive moiety at itsn-terminus or at one or all of the lysine residues K12, K31, and K34. Tomake the modification reaction specific for a particular lysine residue,K12, K31 or K34 can be changed to a non-lysine amino acid, preferablyarginine, and the mutant protein modified with an amine-reactive moietyat the remaining lysine residue. Additionally, all of the lysineresidues, K12, K31, and K34, can be changed to non-lysine residues,preferably arginine residues, and a new lysine residue introduced intothe protein. The new lysine residue can be added preceding the firstamino acid, H1, or following the last amino acid A38. A lysine residuealso can be introduced into the protein by substitution for at least oneamino acid selected from the group consisting of H1, S2, Q3, G4, T5, F6,T7, S8, D9, Y10, S11, Y13, L14, D15, S16, R17, R18, A19, Q20, D21, F22,V23, Q24, W25, L26, M27, N28, D29, T30, R32, N33, N35, N36, I37, A38.These amino acids can be made in the context of other OXM analogs, suchas those described in U.S. Pat. No. 5,858,975. The lysine variants ofthe present invention can be modified with amine-reactive moieties suchas amine-reactive PEGs. The amine-modified peptides can be purified fromunmodified peptides by any of a number of methods known in the art suchas column chromatography.

OXM also can be modified at its N-terminus using methods described inthe art. The modified OXM proteins can be tested for in vitro and invivo biological activity using methods well known in the art.

Example 18 GIP

Gastric inhibitory peptide (GIP) is a small 42 amino acid peptidesecreted by the intestine. GIP increases insulin secretion by thepancreas and may prove useful for treating diabetes. The amino acidsequence of GIP is represented herein by SEQ ID NO:16. A cysteineresidue can be added preceding the first amino acid, Y1, or followingthe last amino acid Q42. A cysteine residue also can be introduced intothe protein by substitution for at least one amino acid selected fromthe group consisting of Y1, A2, E3, G4, T5, F6, I7, S8, D9, Y10, S11,I12, A13, M14, D15, K16, I17, H18, Q19, Q20, D21, F22, V23, N24, W25,L26, L27, A28, Q29, K30, G31, K32, K33, N34, D35, W36, K37, H38, N39,I40, T41, and Q42. These amino acids can be made in the context of othernative sequence GIP or GIP agonist and antagonist analogs, such as thosedescribed in U.S. Pat. No. 6,410,508, U.S. Pat. No. 6,921,748 and U.S.Pat. No. 7,091,183 and in Green et al. Curr. Pharm. Des. 2004, v 10,pages 3651-3662; Irwin et al. (2006) Biochem Pharmacol v. 72: pages719-728; Alana et al. (2006) J. Biol. Chem. 281: 16370-16376; Gault etal. (2002) Biochem J. v 367: 913-920; Gault et al. (2003) J.Endocrinology v. 176: pages 133-141. A preferred analog is one thatprevents cleavage of Y1 and A2 from the peptide by proteases. Apreferred analog is one in which E3 is substituted with a non-glutamicacid amino acid that prevents cleavage of Y1 and A2 from the peptide. Apreferred antagonist analog is one in which E3 is substituted withproline. The cysteine variants of the present invention can be modifiedwith cysteine-reactive moieties such as cysteine reactive PEGs. Thecysteine-modified peptides can be purified from unmodified peptides byany of a number of methods known in the art such as columnchromatography.

Alternatively, GIP can be modified with an amine-reactive moiety at itsN-terminus or at one or all of the lysine residues K16, K30, K32, K33and K37. To make the modification reaction specific for a particularlysine residue, K16, K30, K32, K33 or K37 can be changed to a non-lysineamino acid, preferably arginine, and the mutant protein modified with anamine-reactive moiety at the remaining lysine residue. Additionally, allof the lysine residues, K16, K30, K32, K33 and K37 can be changed tonon-lysine residues, preferably arginine residues, and a new lysineresidue introduced into the protein. The new lysine residue can be addedpreceding the first amino acid, Y1, or following the last amino acidQ42. A lysine residue also can be introduced into the protein bysubstitution for at least one amino acid selected from the groupconsisting of Y1, A2, E3, G4, T5, F6, I7, S8, D9, Y10, 511, I12, A13,M14, D15, I17, H18, Q19, Q20, D21, F22, V23, N24, W25, L26, L27, A28,Q29, G31, N34, D35, W36, H38, N39, I40, T41, and Q42. These amino acidscan be made in the context of native sequence GIP or GIP agonist andantagonist analogs, such as those described in U.S. Pat. No. 6,410,508,U.S. Pat. No. 6,921,748 and U.S. Pat. No. 7,091,183 and in Green et al.Curr. Pharm. Des. 2004, v 10, pages 3651-3662; Irwin et al. (2006)Biochem Pharmacol v. 72: pages 719-728; Alana et al. (2006) J. Biol.Chem. 281: 16370-16376; Gault et al. (2002) Biochem J. v 367: 913-920;Gault et al. (2003) J. Endocrinology v. 176: pages 133-141. The lysinevariants of the present invention can be modified with amine-reactivemoieties such as amine-reactive PEGs. The amine-modified peptides can bepurified from unmodified peptides by any of a number of methods known inthe art such as column chromatography.

GIP also can be modified at its N-terminus using methods described inthe art. The modified GIP proteins can be tested for in vitro and invivo biological activity using methods well known in the art.

Example 19 Amylin

Amylin is a 37 amino acid peptide that is cosecreted with insulin by thepancreas in response to food intake. Amylin plays a role in maintainingproper glucose levels by inhibiting glucagon secretion under conditionsof hyperglycemia. Amylin also slows gastric emptying and promotessatiety, and thus may play a role in feeding and nutrition disorders.The amino acid sequence of amylin is represented herein by SEQ ID NO:17.Amylin contains two native cysteine at positions 2 and 7. These twocysteines form a disulfide bond. The carboxy-terminal tyrosine residuenormally is amidated. A cysteine residue can be added preceding thefirst amino acid, K1, or following the last amino acid Y37. A cysteineresidue also can be introduced into the protein by substitution for atleast one amino acid selected from the group consisting of K1, N3, T4,A5, T6, A8, T9, Q10, R11, L12, A13, N14, F15, L16, V17, H18, S19, S20,N21, N22, F23, G24, A25, I26, L27, S28, S29, T30, N31, V32, G33, S34,N35, T36, Y37. These amino acids can be made in the context of otheramylin analogs, such as those described in U.S. Pat. No. 5,367,052, U.S.Pat. No. 5,686,411 U.S. Pat. No. 6,087,334, and U.S. Pat. No. 6,610,824.The cysteine variants of the present invention can be modified withcysteine-reactive moieties such as cysteine reactive PEGs. Thecysteine-modified peptides can be purified from unmodified peptides byany of a number of methods known in the art such as columnchromatography.

Alternatively, amylin can be modified with an amine-reactive moiety atits N-terminus K1. A lysine residue can be added preceding the firstamino acid, K1, or following the last amino acid Y37. A lysine residuealso can be introduced into the protein by substitution for at least oneamino acid selected from the group consisting of C2, N3, T4, A5, T6, C7,A8, T9, Q10, R11, L12, A13, N14, F15, L16, V17, H18, S19, S20, N21, N22,F23, G24, A25, I26, L27, S28, S29, T30, N31, V32, G33, S34, N35, T36,Y37. These amino acids can be made in the context of native sequenceamylin or amylin analogs, such as those described in U.S. Pat. No.5,367,052, U.S. Pat. No. 5,686,411 U.S. Pat. No. 6,087,334, and U.S.Pat. No. 6,610,824. These amino acid changes can be made in the contextof native sequence amylin or a variant in which K1 is changed to anon-lysine amino acid, preferably arginine. The lysine variants of thepresent invention can be modified with amine-reactive moieties such asamine-reactive PEGs. The lysine-modified peptides can be purified fromunmodified peptides by any of a number of methods known in the art suchas column chromatography.

The peptides of the preceding Examples 11-19 can be synthesized bychemical methods or expressed as recombinant proteins using recombinantDNA technology. Methods for expressing and/or refolding proteinscontaining added or unpaired cysteine residues are described in U.S.Pat. Nos. 6,753,165 and 7,153,943 and 7,306,931 and in U.S. PatentPublication Nos. PCT/US01/16088 and US 20070111240, all of which areincorporated herein by reference. In a preferred embodiment, bioactivepeptides having free cysteines and lacking essential disulfides arerefolded using the novel method of refolding a protein or peptide asdescribed in detail herein.

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Each publication cited herein is incorporated herein by reference in itsentirety.

Each of the following applications is incorporated herein by referencein its entirety: U.S. Provisional Application Ser. No. 60/870,022, filedDec. 14, 2006; U.S. patent application Ser. No. 10/773,530, filed Feb.5, 2004; U.S. Pat. No. 7,148,333; U.S. Pat. No. 6,608,183; PCTApplication Serial No. PCT/US98/14497, filed Jul. 13, 1998; and U.S.Provisional Application Ser. No. 60/052,S16, filed Jul. 14, 1997.

While various embodiments of the present invention have been describedin detail, it is apparent that modifications and adaptations of thoseembodiments will occur to those skilled in the art. It is to beexpressly understood, however, that such modifications and adaptationsare within the scope of the present invention, as set forth above and inthe following claims.

1-125. (canceled)
 126. An isolated cysteine variant of humaninterferon-gamma (IFN-γ) (SEQ ID NO:1), wherein a cysteine residue issubstituted for at least one amino acid located in at least one regionof IFN-gamma selected from the group consisting of: the A-B loop, theB-C loop, the C-D loop, the D-E loop, the E-F loop, the region precedingHelix A and the region following helix F; and wherein Q1 is deleted orsubstituted by a non-glutamine amino acid.
 127. The cysteine variant ofclaim 126, wherein a cysteine residue is substituted for at least oneamino acid located in the A-B loop.
 128. The cysteine variant of claim126, wherein a cysteine residue is substituted for at least one aminoacid located in the B-C loop.
 129. The cysteine variant of claim 126,wherein a cysteine residue is substituted for at least one amino acidlocated in the C-D loop.
 130. The cysteine variant of claim 126, whereina cysteine residue is substituted for at least one amino acid located inthe D-E loop.
 131. The cysteine variant of claim 126, wherein a cysteineresidue is substituted for at least one amino acid located in the E-Floop.
 132. The cysteine variant of claim 126, wherein a cysteine residueis substituted for at least one amino acid located in the regionpreceding Helix A.
 133. The cysteine variant of claim 126, wherein acysteine residue is substituted for at least one amino acid located inthe region following Helix F.
 134. The cysteine variant of claim 126,wherein Q1 is deleted.
 135. The cysteine variant of claim 126, whereinQ1 is substituted by a non-glutamine amino acid.
 136. The cysteinevariant of claim 131, wherein a cysteine residue is substituted for anamino acid located in the E-F loop selected from the group consisting ofV99, V100, T101, D102, and L103.
 137. The cysteine varinant of claim131, wherein a cysteine residue is substituted for L103.
 138. Thecysteine variant of claim 129, wherein a cysteine residue is substitutedfor an amino acid located in the C-D loop selected from the groupconsisting of D63, Q64, S65, I66, and Q67.
 139. The cysteine variant ofclaim 129, wherein a cysteine residue is substituted for Q67.
 140. Thecysteine variant of claim 126, further comprising substitution of the D2amino acid in the region preceding Helix A with a non-aspartic acid.141. The cysteine variant of claim 126, further comprising deletion ofthe D2 amino acid in the region preceding Helix A.
 142. The cysteinevariant of claim 126 wherein said variant is modified with a cysteinereactive moiety.
 143. The cysteine variant of claim 142, wherein saidcysteine reactive moiety is a polyethylene glycol.
 144. The cysteinevariant of claim 126, wherein said variant is a recombinant protein.145. A method for treating a patient with a disease treatable withIFN-gamma, comprising administering to said patient a therapeuticallyeffective amount of a cysteine variant of IFN-gamma of claim 126.